Compositions for use in identification of bacteria

ABSTRACT

The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/409,535, filed Apr. 21, 2006, which is a continuation-in-part of U.S.application Ser. No. 11/060,135, filed Feb. 17, 2005 which claims thebenefit of priority to U.S. Provisional Application Ser. No. 60/545,425filed Feb. 18, 2004; U.S. Provisional Application Ser. No. 60/559,754,filed Apr. 5, 2004; U.S. Provisional Application Ser. No. 60/632,862,filed Dec. 3, 2004; U.S. Provisional Application Ser. No. 60/639,068,filed Dec. 22, 2004; and U.S. Provisional Application Ser. No.60/648,188, filed Jan. 28, 2005. U.S. application Ser. No. 11/409,535 isa also continuation-in-part of U.S. application Ser. No. 10/728,486,filed Dec. 5, 2003 which claims the benefit of priority to U.S.Provisional Application Ser. No. 60/501,926, filed Sep. 11, 2003. U.S.application Ser. No. 11/409,535 also claims the benefit of priority to:U.S. Provisional Application Ser. No. 60/674,118, filed Apr. 21, 2005;U.S. Provisional Application Ser. No. 60/705,631, filed Aug. 3, 2005;U.S. Provisional Application Ser. No. 60/732,539, filed Nov. 1, 2005;and U.S. Provisional Application Ser. No. 60/773,124, filed Feb. 13,2006. Each of the above-referenced U.S. applications is incorporatedherein by reference in its entirety. Methods disclosed in U.S.application Ser. Nos. 09/891,793, 10/156,608, 10/405,756, 10/418,514,10/660,122, 10,660,996, 10/660,997, 10/660,998, 10/728,486, 11/060,135,and 11/073,362, are commonly owned and incorporated herein by referencein their entirety for any purpose.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States Government support under CDCcontract RO1 CI000099-01. The United States Government has certainrights in the invention.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledDIBIS0083USC13SEQ.txt, created on Mar. 13, 2007 which is 252 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions, kits and methods for rapididentification and quantification of bacteria by molecular mass and basecomposition analysis.

BACKGROUND OF THE INVENTION

A problem in determining the cause of a natural infectious outbreak or abioterrorist attack is the sheer variety of organisms that can causehuman disease. There are over 1400 organisms infectious to humans; manyof these have the potential to emerge suddenly in a natural epidemic orto be used in a malicious attack by bioterrorists (Taylor et al. Philos.Trans. R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This numberdoes not include numerous strain variants, bioengineered versions, orpathogens that infect plants or animals.

Much of the new technology being developed for detection of biologicalweapons incorporates a polymerase chain reaction (PCR) step based uponthe use of highly specific primers and probes designed to selectivelydetect certain pathogenic organisms. Although this approach isappropriate for the most obvious bioterrorist organisms, like smallpoxand anthrax, experience has shown that it is very difficult to predictwhich of hundreds of possible pathogenic organisms might be employed ina terrorist attack. Likewise, naturally emerging human disease that hascaused devastating consequence in public health has come from unexpectedfamilies of bacteria, viruses, fungi, or protozoa. Plants and animalsalso have their natural burden of infectious disease agents and thereare equally important biosafety and security concerns for agriculture.

A major conundrum in public health protection, biodefense, andagricultural safety and security is that these disciplines need to beable to rapidly identify and characterize infectious agents, while thereis no existing technology with the breadth of function to meet thisneed. Currently used methods for identification of bacteria rely uponculturing the bacterium to effect isolation from other organisms and toobtain sufficient quantities of nucleic acid followed by sequencing ofthe nucleic acid, both processes which are time and labor intensive.

Mass spectrometry provides detailed information about the moleculesbeing analyzed, including high mass accuracy. It is also a process thatcan be easily automated. DNA chips with specific probes can onlydetermine the presence or absence of specifically anticipated organisms.Because there are hundreds of thousands of species of benign bacteria,some very similar in sequence to threat organisms, even arrays with10,000 probes lack the breadth needed to identify a particular organism.

The present invention provides oligonucleotide primers and compositionsand kits containing the oligonucleotide primers, which define bacterialbioagent identifying amplicons and, upon amplification, producecorresponding amplification products whose molecular masses provide themeans to identify bacteria, for example, at and below the speciestaxonomic level.

SUMMARY OF THE INVENTION

The present invention provides compositions, kits and methods for rapididentification and quantification of bacteria by molecular mass and basecomposition analysis.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 456.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1261.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 456 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1261.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 288.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1269.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 288 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1269.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 698.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1420.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 698 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1420.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 217.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1167

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 217 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1167.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 399.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1041.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 399 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1041.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 430.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1321.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 430 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1321.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 174.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 853.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 174 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 853.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 172.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1360.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 172 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1360.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 456 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1261.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 456 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1261 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 288:1269, 698:1420, 217:1167, 399:1041,430:1321, 174:853, and 172:1360.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 681.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1022.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 681 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1022.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 315.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1379.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 315 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1379.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 346.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 955.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 346 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 955.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 504.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1409.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 504 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1409.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 323.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1068.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 323 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 1068.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 479.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 938.

Another embodiment is an oligonucleotide primer pair including anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 479 and an oligonucleotide primer14 to 35 nucleobases in length having at least 70% sequence identitywith SEQ ID NO: 938.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 681 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1022.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 681 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1022 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 315:1379, 346:955, 504:1409, 323:1068,479:938.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 583.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 923.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 583 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 923.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 454.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1418.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 454 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1418.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 250.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 902.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 250 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 902.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 384.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 878.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 384 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 878.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 694.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1215.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 694 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1215.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 194.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1173.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 194 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1173.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 375.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 890.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 375 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 890.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 656.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1224.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 656 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1224.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 618.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1157.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 618 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1157.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 302.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 852.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 302 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 852.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 199.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 889.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 199 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 889.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 596.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1169.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 596 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1169.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 150.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1242.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 150 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1242.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 166.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1069.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 166 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1069.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 166.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1168.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 166 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1168.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 583 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 923 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 454:1418, 250:902, 384:878, 694:1215,194:1173, 375:890, 656:1224, 618:1157, 302:852, 199:889, 596:1169,150:1242, 166:1069 and 166:1168.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 437.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1137.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 437 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1137.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 530.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 891.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 530 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 891.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 474.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 869.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 474 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 869.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 268.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1284.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 268 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1284.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 418.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1301.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 418 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1301.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 318.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1300.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 318 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1300.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 440.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1076.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 440 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1076.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 219.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1013.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 219 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1013.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 437 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1137 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 530:891, 474:869, 268:1284, 418:1301,318:1300, 440:1076 and 219:1013.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 325.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1163.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 325 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1163.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 278.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1039.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 278 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1039.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 465.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1037.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 465 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1037.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 148.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1172.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 148 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1172.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 190.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1254.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 190 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1254.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 266.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1094.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 266 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1094.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 508.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1297.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 508 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1297.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 259.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1060.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 259 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1060.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 325 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1163 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 278:1039: 465:1037, 148:1172, 190:1254,266:1094, 508:1297 and 259:1060.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 376.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1265.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 376 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1265.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 267.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1341.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 267 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1341.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 705.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1056.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 705 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1056.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 710.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1259.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 710 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1259.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 374.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1111.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 374 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1111.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 545.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 978.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 545 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 978.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 249.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1095.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 249 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1095.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 195.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1376.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 195 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1376.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 311.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1014.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 311 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1014.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 365.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1052.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 365 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1052.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 527.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1071.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 527 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1071.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 490.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases inlength having at least 70% sequence identity with SEQ ID NO: 1182.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 490 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1182.

Another embodiment is a kit comprising an oligonucleotide primer pairincluding an oligonucleotide primer 14 to 35 nucleobases in lengthhaving at least 70% sequence identity with SEQ ID NO: 376 and anoligonucleotide primer 14 to 35 nucleobases in length having at least70% sequence identity with SEQ ID NO: 1265 and further comprising one ormore primer pairs wherein each member of said one or more primer pairsis of a length of 14 to 35 nucleobases and has 70% to 100% sequenceidentity with the corresponding member from the group of primer pairsrepresented by SEQ ID NOs: 267:1341, 705:1056, 710:1259, 374:1111,545:978, 249:1095, 195:1376, 311:1014, 365:1052, 527:1071 and 490:1182.

In some embodiments, either or both of the primers of a primer paircomposition contain at least one modified nucleobase such as5-propynyluracil or 5-propynylcytosine for example.

In some embodiments, either or both of the primers of the primer paircomprises at least one universal nucleobase such as inosine for example.

In some embodiments, either or both of the primers of the primer paircomprises at least one non-templated T residue on the 5′-end.

In some embodiments, either or both of the primers of the primer paircomprises at least one non-template tag.

In some embodiments, either or both of the primers of the primer paircomprises at least one molecular mass modifying tag.

In some embodiments, the present invention provides primers andcompositions comprising pairs of primers, and kits containing the same,and methods for use in identification of bacteria. The primers aredesigned to produce amplification products of DNA encoding genes thathave conserved and variable regions across different subgroups andgenotypes of bacteria.

Some embodiments are kits that contain one or more of the primer paircompositions. In some embodiments, each member of the one or more primerpairs of the kit is of a length of 14 to 35 nucleobases and has 70% to100% sequence identity with the corresponding member from any of theprimer pairs listed in Table 2.

Some embodiments of the kits contain at least one calibrationpolynucleotide for use in quantitation of bacteria in a given sample,and also for use as a positive control for amplification.

Some embodiments of the kits contain at least one anion exchangefunctional group linked to a magnetic bead.

In some embodiments, the present invention also provides methods foridentification of bacteria. Nucleic acid from the bacterium is amplifiedusing the primers described above to obtain an amplification product.The molecular mass of the amplification product is measured. Optionally,the base composition of the amplification product is determined from themolecular mass. The molecular mass or base composition is compared witha plurality of molecular masses or base compositions of known analogousbacterial identifying amplicons, wherein a match between the molecularmass or base composition and a member of the plurality of molecularmasses or base compositions identifies the bacterium. In someembodiments, the molecular mass is measured by mass spectrometry in amodality such as electrospray ionization (ESI) time of flight (TOF) massspectrometry or ESI Fourier transform ion cyclotron resonance (FTICR)mass spectrometry, for example. Other mass spectrometry techniques canalso be used to measure the molecular mass of bacterial bioagentidentifying amplicons.

In some embodiments, the present invention is also directed to a methodfor determining the presence or absence of a bacterium in a sample.Nucleic acid from the sample is amplified using the compositiondescribed above to obtain an amplification product. The molecular massof the amplification product is determined. Optionally, the basecomposition of the amplification product is determined from themolecular mass. The molecular mass or base composition of theamplification product is compared with the known molecular masses orbase compositions of one or more known analogous bacterial bioagentidentifying amplicons, wherein a match between the molecular mass orbase composition of the amplification product and the molecular mass orbase composition of one or more known bacterial bioagent identifyingamplicons indicates the presence of the bacterium in the sample. In someembodiments, the molecular mass is measured by mass spectrometry.

In some embodiments, the present invention also provides methods fordetermination of the quantity of an unknown bacterium in a sample. Thesample is contacted with the composition described above and a knownquantity of a calibration polynucleotide comprising a calibrationsequence. Nucleic acid from the unknown bacterium in the sample isconcurrently amplified with the composition described above and nucleicacid from the calibration polynucleotide in the sample is concurrentlyamplified with the composition described above to obtain a firstamplification product comprising a bacterial bioagent identifyingamplicon and a second amplification product comprising a calibrationamplicon. The molecular masses and abundances for the bacterial bioagentidentifying amplicon and the calibration amplicon are determined. Thebacterial bioagent identifying amplicon is distinguished from thecalibration amplicon based on molecular mass and comparison of bacterialbioagent identifying amplicon abundance and calibration ampliconabundance indicates the quantity of bacterium in the sample. In someembodiments, the base composition of the bacterial bioagent identifyingamplicon is determined.

In some embodiments, the present invention provides methods fordetecting or quantifying bacteria by combining a nucleic acidamplification process with a mass determination process. In someembodiments, such methods identify or otherwise analyze the bacterium bycomparing mass information from an amplification product with acalibration or control product. Such methods can be carried out in ahighly multiplexed and/or parallel manner allowing for the analysis ofas many as 300 samples per 24 hours on a single mass measurementplatform. The accuracy of the mass determination methods in someembodiments of the present invention permits allows for the ability todiscriminate between different bacteria such as, for example, variousgenotypes and drug resistant strains of Staphylococcus aureus.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary of the invention, as well as the followingdetailed description of the invention, is better understood when read inconjunction with the accompanying drawings which are included by way ofexample and not by way of limitation.

FIG. 1: process diagram illustrating a representative primer pairselection process.

FIG. 2: process diagram illustrating an embodiment of the calibrationmethod.

FIG. 3: common pathogenic bacteria and primer pair coverage. The primerpair number in the upper right hand corner of each polygon indicatesthat the primer pair can produce a bioagent identifying amplicon for allspecies within that polygon.

FIG. 4: a representative 3D diagram of base composition (axes A, G andC) of bioagent identifying amplicons obtained with primer pair number 14(a precursor of primer pair number 348 which targets 16S rRNA). Thediagram indicates that the experimentally determined base compositionsof the clinical samples (labeled NHRC samples) closely match the basecompositions expected for Streptococcus pyogenes and are distinct fromthe expected base compositions of other organisms.

FIG. 5: a representative mass spectrum of amplification productsindicating the presence of bioagent identifying amplicons ofStreptococcus pyogenes, Neisseria meningitidis, and Haemophilusinfluenzae obtained from amplification of nucleic acid from a clinicalsample with primer pair number 349 which targets 23S rRNA.Experimentally determined molecular masses and base compositions for thesense strand of each amplification product are shown.

FIG. 6: a representative mass spectrum of amplification productsrepresenting a bioagent identifying amplicon of Streptococcus pyogenes,and a calibration amplicon obtained from amplification of nucleic acidfrom a clinical sample with primer pair number 356 which targets rplB.The experimentally determined molecular mass and base composition forthe sense strand of the Streptococcus pyogenes amplification product isshown.

FIG. 7: a representative mass spectrum of an amplified nucleic acidmixture which contained the Ames strain of Bacillus anthracis, a knownquantity of combination calibration polynucleotide (SEQ ID NO: 1464),and primer pair number 350 which targets the capC gene on the virulenceplasmid pX02 of Bacillus anthracis. Calibration amplicons produced inthe amplification reaction are visible in the mass spectrum as indicatedand abundance data (peak height) are used to calculate the quantity ofthe Ames strain of Bacillus anthracis.

DEFINITIONS

As used herein, the term “abundance” refers to an amount. The amount maybe described in terms of concentration which are common in molecularbiology such as “copy number,” “pfu or plate-forming unit” which arewell known to those with ordinary skill. Concentration may be relativeto a known standard or may be absolute.

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” also comprises “sampletemplate.”

As used herein the term “amplification” refers to a special case ofnucleic acid replication involving template specificity. It is to becontrasted with non-specific template replication (i.e., replicationthat is template-dependent but not dependent on a specific template).Template specificity is here distinguished from fidelity of replication(i.e., synthesis of the proper polynucleotide sequence) and nucleotide(ribo- or deoxyribo-) specificity. Template specificity is frequentlydescribed in terms of “target” specificity. Target sequences are“targets” in the sense that they are sought to be sorted out from othernucleic acid. Amplification techniques have been designed primarily forthis sorting out. Template specificity is achieved in most amplificationtechniques by the choice of enzyme. Amplification enzymes are enzymesthat, under conditions they are used, will process only specificsequences of nucleic acid in a heterogeneous mixture of nucleic acid.For example, in the case of Qβ replicase, MDV-1 RNA is the specifictemplate for the replicase (D. L. Kacian et al., Proc. Natl. Acad. Sci.USA 69:3038 [1972]). Other nucleic acid will not be replicated by thisamplification enzyme. Similarly, in the case of T7 RNA polymerase, thisamplification enzyme has a stringent specificity for its own promoters(Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNAligase, the enzyme will not ligate the two oligonucleotides orpolynucleotides, where there is a mismatch between the oligonucleotideor polynucleotide substrate and the template at the ligation junction(D. Y. Wu and R. B. Wallace, Genomics 4:560 [1989]). Finally, Taq andPfu polymerases, by virtue of their ability to function at hightemperature, are found to display high specificity for the sequencesbounded and thus defined by the primers; the high temperature results inthermodynamic conditions that favor primer hybridization with the targetsequences and not hybridization with non-target sequences (H. A. Erlich(ed.), PCR Technology, Stockton Press [1989]).

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification, excluding primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the term “analogous” when used in context of comparisonof bioagent identifying amplicons indicates that the bioagentidentifying amplicons being compared are produced with the same pair ofprimers. For example, bioagent identifying amplicon “A” and bioagentidentifying amplicon “B”, produced with the same pair of primers areanalogous with respect to each other. Bioagent identifying amplicon “C”,produced with a different pair of primers is not analogous to eitherbioagent identifying amplicon “A” or bioagent identifying amplicon “B”.

As used herein, the term “anion exchange functional group” refers to apositively charged functional group capable of binding an anion throughan electrostatic interaction. The most well known anion exchangefunctional groups are the amines, including primary, secondary, tertiaryand quaternary amines.

The term “bacteria” or “bacterium” refers to any member of the groups ofeubacteria and archaebacteria.

As used herein, a “base composition” is the exact number of eachnucleobase (for example, A, T, C and G) in a segment of nucleic acid.For example, amplification of nucleic acid of Staphylococcus aureusstrain carrying the lukS-PV gene with primer pair number 2095 (SEQ IDNOs: 456:1261) produces an amplification product 117 nucleobases inlength from nucleic acid of the lukS-PV gene that has a base compositionof A35 G17 C19 T46 (by convention—with reference to the sense strand ofthe amplification product). Because the molecular masses of each of thefour natural nucleotides and chemical modifications thereof are known(if applicable), a measured molecular mass can be deconvoluted to a listof possible base compositions. Identification of a base composition of asense strand which is complementary to the corresponding antisensestrand in terms of base composition provides a confirmation of the truebase composition of an unknown amplification product. For example, thebase composition of the antisense strand of the 139 nucleobaseamplification product described above is A46 G19 C17 T35.

As used herein, a “base composition probability cloud” is arepresentation of the diversity in base composition resulting from avariation in sequence that occurs among different isolates of a givenspecies. The “base composition probability cloud” represents the basecomposition constraints for each species and is typically visualizedusing a pseudo four-dimensional plot.

In the context of this invention, a “bioagent” is any organism, cell, orvirus, living or dead, or a nucleic acid derived from such an organism,cell or virus. Examples of bioagents include, but are not limited, tocells, (including but not limited to human clinical samples, bacterialcells and other pathogens), viruses, fungi, protists, parasites, andpathogenicity markers (including but not limited to: pathogenicityislands, antibiotic resistance genes, virulence factors, toxin genes andother bioregulating compounds). Samples may be alive or dead or in avegetative state (for example, vegetative bacteria or spores) and may beencapsulated or bioengineered. In the context of this invention, a“pathogen” is a bioagent which causes a disease or disorder.

As used herein, a “bioagent division” is defined as group of bioagentsabove the species level and includes but is not limited to, orders,families, classes, clades, genera or other such groupings of bioagentsabove the species level.

As used herein, the term “bioagent identifying amplicon” refers to apolynucleotide that is amplified from a bioagent in an amplificationreaction and which 1) provides sufficient variability to distinguishamong bioagents from whose nucleic acid the bioagent identifyingamplicon is produced and 2) whose molecular mass is amenable to a rapidand convenient molecular mass determination modality such as massspectrometry, for example.

As used herein, the term “biological product” refers to any productoriginating from an organism. Biological products are often products ofprocesses of biotechnology. Examples of biological products include, butare not limited to: cultured cell lines, cellular components,antibodies, proteins and other cell-derived biomolecules, growth media,growth harvest fluids, natural products and bio-pharmaceutical products.

The terms “biowarfare agent” and “bioweapon” are synonymous and refer toa bacterium, virus, fungus or protozoan that could be deployed as aweapon to cause bodily harm to individuals. Military or terrorist groupsmay be implicated in deployment of biowarfare agents.

In context of this invention, the term “broad range survey primer pair”refers to a primer pair designed to produce bioagent identifyingamplicons across different broad groupings of bioagents. For example,the ribosomal RNA-targeted primer pairs are broad range survey primerpairs which have the capability of producing bacterial bioagentidentifying amplicons for essentially all known bacteria. With respectto broad range primer pairs employed for identification of bacteria, abroad range survey primer pair for bacteria such as 16S rRNA primer pairnumber 346 (SEQ ID NOs: 202:1110) for example, will produce an bacterialbioagent identifying amplicon for essentially all known bacteria.

The term “calibration amplicon” refers to a nucleic acid segmentrepresenting an amplification product obtained by amplification of acalibration sequence with a pair of primers designed to produce abioagent identifying amplicon.

The term “calibration sequence” refers to a polynucleotide sequence towhich a given pair of primers hybridizes for the purpose of producing aninternal (i.e: included in the reaction) calibration standardamplification product for use in determining the quantity of a bioagentin a sample. The calibration sequence may be expressly added to anamplification reaction, or may already be present in the sample prior toanalysis.

The term “clade primer pair” refers to a primer pair designed to producebioagent identifying amplicons for species belonging to a clade group. Aclade primer pair may also be considered as a “speciating” primer pairwhich is useful for distinguishing among closely related species.

The term “codon” refers to a set of three adjoined nucleotides (triplet)that codes for an amino acid or a termination signal.

In context of this invention, the term “codon base compositionanalysis,” refers to determination of the base composition of anindividual codon by obtaining a bioagent identifying amplicon thatincludes the codon. The bioagent identifying amplicon will at leastinclude regions of the target nucleic acid sequence to which the primershybridize for generation of the bioagent identifying amplicon as well asthe codon being analyzed, located between the two primer hybridizationregions.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides such asan oligonucleotide or a target nucleic acid) related by the base-pairingrules. For example, for the sequence “5′-A-G-T-3′,” is complementary tothe sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in whichonly some of the nucleic acids' bases are matched according to the basepairing rules. Or, there may be “complete” or “total” complementaritybetween the nucleic acids. The degree of complementarity between nucleicacid strands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methods thatdepend upon binding between nucleic acids. Either term may also be usedin reference to individual nucleotides, especially within the context ofpolynucleotides. For example, a particular nucleotide within anoligonucleotide may be noted for its complementarity, or lack thereof,to a nucleotide within another nucleic acid strand, in contrast orcomparison to the complementarity between the rest of theoligonucleotide and the nucleic acid strand.

The term “complement of a nucleic acid sequence” as used herein refersto an oligonucleotide which, when aligned with the nucleic acid sequencesuch that the 5′ end of one sequence is paired with the 3′ end of theother, is in “antiparallel association.” Certain bases not commonlyfound in natural nucleic acids may be included in the nucleic acids ofthe present invention and include, for example, inosine and7-deazaguanine. Complementarity need not be perfect; stable duplexes maycontain mismatched base pairs or unmatched bases. Those skilled in theart of nucleic acid technology can determine duplex stabilityempirically considering a number of variables including, for example,the length of the oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.Where a first oligonucleotide is complementary to a region of a targetnucleic acid and a second oligonucleotide has complementary to the sameregion (or a portion of this region) a “region of overlap” exists alongthe target nucleic acid. The degree of overlap will vary depending uponthe extent of the complementarity.

In context of this invention, the term “division-wide primer pair”refers to a primer pair designed to produce bioagent identifyingamplicons within sections of a broader spectrum of bioagents Forexample, primer pair number 352 (SEQ ID NOs: 687:1411), a division-wideprimer pair, is designed to produce bacterial bioagent identifyingamplicons for members of the Bacillus group of bacteria which comprises,for example, members of the genera Streptococci, Enterococci, andStaphylococci. Other division-wide primer pairs may be used to producebacterial bioagent identifying amplicons for other groups of bacterialbioagents.

As used herein, the term “concurrently amplifying” used with respect tomore than one amplification reaction refers to the act of simultaneouslyamplifying more than one nucleic acid in a single reaction mixture.

As used herein, the term “drill-down primer pair” refers to a primerpair designed to produce bioagent identifying amplicons foridentification of sub-species characteristics or confirmation of aspecies assignment. For example, primer pair number 2146 (SEQ ID NOs:437:1137), a drill-down Staphylococcus aureus genotyping primer pair, isdesigned to produce Staphylococcus aureus genotyping amplicons. Otherdrill-down primer pairs may be used to produce bioagent identifyingamplicons for Staphylococcus aureus and other bacterial species.

The term “duplex” refers to the state of nucleic acids in which the baseportions of the nucleotides on one strand are bound through hydrogenbonding the their complementary bases arrayed on a second strand. Thecondition of being in a duplex form reflects on the state of the basesof a nucleic acid. By virtue of base pairing, the strands of nucleicacid also generally assume the tertiary structure of a double helix,having a major and a minor groove. The assumption of the helical form isimplicit in the act of becoming duplexed.

As used herein, the term “etiology” refers to the causes or origins, ofdiseases or abnormal physiological conditions.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of an RNA having anon-coding function (e.g., a ribosomal or transfer RNA), a polypeptideor a precursor. The RNA or polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity or function is retained.

The terms “homology,” “homologous” and “sequence identity” refer to adegree of identity. There may be partial homology or complete homology.A partially homologous sequence is one that is less than 100% identicalto another sequence. Determination of sequence identity is described inthe following example: a primer 20 nucleobases in length which isotherwise identical to another 20 nucleobase primer but having twonon-identical residues has 18 of 20 identical residues (18/20=0.9 or 90%sequence identity). In another example, a primer 15 nucleobases inlength having all residues identical to a 15 nucleobase segment of aprimer 20 nucleobases in length would have 15/20=0.75 or 75% sequenceidentity with the 20 nucleobase primer. In context of the presentinvention, sequence identity is meant to be properly determined when thequery sequence and the subject sequence are both described and alignedin the 5′ to 3′ direction. Sequence alignment algorithms such as BLAST,will return results in two different alignment orientations. In thePlus/Plus orientation, both the query sequence and the subject sequenceare aligned in the 5′ to 3′ direction. On the other hand, in thePlus/Minus orientation, the query sequence is in the 5′ to 3′ directionwhile the subject sequence is in the 3′ to 5′ direction. It should beunderstood that with respect to the primers of the present invention,sequence identity is properly determined when the alignment isdesignated as Plus/Plus. Sequence identity may also encompass alternateor modified nucleobases that perform in a functionally similar manner tothe regular nucleobases adenine, thymine, guanine and cytosine withrespect to hybridization and primer extension in amplificationreactions. In a non-limiting example, if the 5-propynyl pyrimidinespropyne C and/or propyne T replace one or more C or T residues in oneprimer which is otherwise identical to another primer in sequence andlength, the two primers will have 100% sequence identity with eachother. In another non-limiting example, Inosine (I) may be used as areplacement for G or T and effectively hybridize to C, A or U (uracil).Thus, if inosine replaces one or more C, A or U residues in one primerwhich is otherwise identical to another primer in sequence and length,the two primers will have 100% sequence identity with each other. Othersuch modified or universal bases may exist which would perform in afunctionally similar manner for hybridization and amplificationreactions and will be understood to fall within this definition ofsequence identity.

As used herein, “housekeeping gene” refers to a gene encoding a proteinor RNA involved in basic functions required for survival andreproduction of a bioagent. Housekeeping genes include, but are notlimited to genes encoding RNA or proteins involved in translation,replication, recombination and repair, transcription, nucleotidemetabolism, amino acid metabolism, lipid metabolism, energy generation,uptake, secretion and the like.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is influenced by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, and the T_(m) of the formed hybrid. “Hybridization” methodsinvolve the annealing of one nucleic acid to another, complementarynucleic acid, i.e., a nucleic acid having a complementary nucleotidesequence. The ability of two polymers of nucleic acid containingcomplementary sequences to find each other and anneal through basepairing interaction is a well-recognized phenomenon. The initialobservations of the “hybridization” process by Marmur and Lane, Proc.Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad.Sci. USA 46:461 (1960) have been followed by the refinement of thisprocess into an essential tool of modem biology.

The term “in silico” refers to processes taking place via computercalculations. For example, electronic PCR (ePCR) is a process analogousto ordinary PCR except that it is carried out using nucleic acidsequences and primer pair sequences stored on a computer formattedmedium.

As used herein, “intelligent primers” are primers that are designed tobind to highly conserved sequence regions of a bioagent identifyingamplicon that flank an intervening variable region and, uponamplification, yield amplification products which ideally provide enoughvariability to distinguish individual bioagents, and which are amenableto molecular mass analysis. By the term “highly conserved,” it is meantthat the sequence regions exhibit between about 80-100%, or betweenabout 90-100%, or between about 95-100% identity among all, or at least70%, at least 80%, at least 90%, at least 95%, or at least 99% ofspecies or strains.

The “ligase chain reaction” (LCR; sometimes referred to as “LigaseAmplification Reaction” (LAR) described by Barany, Proc. Natl. Acad.Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wuand Wallace, Genomics 4:560 (1989) has developed into a well-recognizedalternative method for amplifying nucleic acids. In LCR, fouroligonucleotides, two adjacent oligonucleotides which uniquely hybridizeto one strand of target DNA, and a complementary set of adjacentoligonucleotides, that hybridize to the opposite strand are mixed andDNA ligase is added to the mixture. Provided that there is completecomplementarity at the junction, ligase will covalently link each set ofhybridized molecules. Importantly, in LCR, two probes are ligatedtogether only when they base-pair with sequences in the target sample,without gaps or mismatches. Repeated cycles of denaturation,hybridization and ligation amplify a short segment of DNA. LCR has alsobeen used in combination with PCR to achieve enhanced detection ofsingle-base changes. However, because the four oligonucleotides used inthis assay can pair to form two short ligatable fragments, there is thepotential for the generation of target-independent background signal.The use of LCR for mutant screening is limited to the examination ofspecific nucleic acid positions.

The term “locked nucleic acid” or “LNA” refers to a nucleic acidanalogue containing one or more 2′-O, 4′-C-methylene-β-D-ribofuranosylnucleotide monomers in an RNA mimicking sugar conformation. LNAoligonucleotides display unprecedented hybridization affinity towardcomplementary single-stranded RNA and complementary single- ordouble-stranded DNA. LNA oligonucleotides induce A-type (RNA-like)duplex conformations. The primers of the present invention may containLNA modifications.

As used herein, the term “mass-modifying tag” refers to any modificationto a given nucleotide which results in an increase in mass relative tothe analogous non-mass modified nucleotide. Mass-modifying tags caninclude heavy isotopes of one or more elements included in thenucleotide such as carbon-13 for example. Other possible modificationsinclude addition of substituents such as iodine or bromine at the 5position of the nucleobase for example.

The term “mass spectrometry” refers to measurement of the mass of atomsor molecules. The molecules are first converted to ions, which areseparated using electric or magnetic fields according to the ratio oftheir mass to electric charge. The measured masses are used to identitythe molecules.

The term “microorganism” as used herein means an organism too small tobe observed with the unaided eye and includes, but is not limited tobacteria, virus, protozoans, fungi; and ciliates.

The term “multi-drug resistant” or multiple-drug resistant” refers to amicroorganism which is resistant to more than one of the antibiotics orantimicrobial agents used in the treatment of said microorganism.

The term “multiplex PCR” refers to a PCR reaction where more than oneprimer set is included in the reaction pool allowing 2 or more differentDNA targets to be amplified by PCR in a single reaction tube.

The term “non-template tag” refers to a stretch of at least threeguanine or cytosine nucleobases of a primer used to produce a bioagentidentifying amplicon which are not complementary to the template. Anon-template tag is incorporated into a primer for the purpose ofincreasing the primer-duplex stability of later cycles of amplificationby incorporation of extra G-C pairs which each have one additionalhydrogen bond relative to an A-T pair.

The term “nucleic acid sequence” as used herein refers to the linearcomposition of the nucleic acid residues A, T, C or G or anymodifications thereof, within an oligonucleotide, nucleotide orpolynucleotide, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin which may be single or double stranded, andrepresent the sense or antisense strand

As used herein, the term “nucleobase” is synonymous with other terms inuse in the art including “nucleotide,” “deoxynucleotide,” “nucleotideresidue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” ordeoxynucleotide triphosphate (dNTP).

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides such as 5-propynyl pyrimidines(i.e., 5-propynyl-dTTP and 5-propynyl-dTCP), 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogsand comprise modified forms of deoxyribonucleotides as well asribonucleotides.

The term “oligonucleotide” as used herein is defined as a moleculecomprising two or more deoxyribonucleotides or ribonucleotides,preferably at least 5 nucleotides, more preferably at least about 13 to35 nucleotides. The exact size will depend on many factors, which inturn depend on the ultimate function or use of the oligonucleotide. Theoligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, PCR, or a combinationthereof. Because mononucleotides are reacted to make oligonucleotides ina manner such that the 5′ phosphate of one mononucleotide pentose ringis attached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′-end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′-end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction. Alloligonucleotide primers disclosed herein are understood to be presentedin the 5′ to 3′ direction when reading left to right. When twodifferent, non-overlapping oligonucleotides anneal to different regionsof the same linear complementary nucleic acid sequence, and the 3′ endof one oligonucleotide points towards the 5′ end of the other, theformer may be called the “upstream” oligonucleotide and the latter the“downstream” oligonucleotide. Similarly, when two overlappingoligonucleotides are hybridized to the same linear complementary nucleicacid sequence, with the first oligonucleotide positioned such that its5′ end is upstream of the 5′ end of the second oligonucleotide, and the3′ end of the first oligonucleotide is upstream of the 3′ end of thesecond oligonucleotide, the first oligonucleotide may be called the“upstream” oligonucleotide and the second oligonucleotide may be calledthe “downstream” oligonucleotide.

In the context of this invention, a “pathogen” is a bioagent whichcauses a disease or disorder.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

The term “peptide nucleic acid” (“PNA”) as used herein refers to amolecule comprising bases or base analogs such as would be found innatural nucleic acid, but attached to a peptide backbone rather than thesugar-phosphate backbone typical of nucleic acids. The attachment of thebases to the peptide is such as to allow the bases to base pair withcomplementary bases of nucleic acid in a manner similar to that of anoligonucleotide. These small molecules, also designated anti geneagents, stop transcript elongation by binding to their complementarystrand of nucleic acid (Nielsen, et al. Anticancer Drug Des. 8:53 63).The primers of the present invention may comprise PNAs.

The term “polymerase” refers to an enzyme having the ability tosynthesize a complementary strand of nucleic acid from a startingtemplate nucleic acid strand and free dNTPs.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and4,965,188, hereby incorporated by reference, that describe a method forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing, and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified.” With PCR, it is possible to amplify a single copy ofa specific target sequence in genomic DNA to a level detectable byseveral different methodologies (e.g., hybridization with a labeledprobe; incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of 32P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide or polynucleotide sequencecan be amplified with the appropriate set of primer molecules. Inparticular, the amplified segments created by the PCR process itselfare, themselves, efficient templates for subsequent PCR amplifications.

The term “polymerization means” or “polymerization agent” refers to anyagent capable of facilitating the addition of nucleoside triphosphatesto an oligonucleotide. Preferred polymerization means comprise DNA andRNA polymerases.

As used herein, the terms “pair of primers,” or “primer pair” aresynonymous. A primer pair is used for amplification of a nucleic acidsequence. A pair of primers comprises a forward primer and a reverseprimer. The forward primer hybridizes to a sense strand of a target genesequence to be amplified and primes synthesis of an antisense strand(complementary to the sense strand) using the target sequence as atemplate. A reverse primer hybridizes to the antisense strand of atarget gene sequence to be amplified and primes synthesis of a sensestrand (complementary to the antisense strand) using the target sequenceas a template.

The primers are designed to bind to highly conserved sequence regions ofa bioagent identifying amplicon that flank an intervening variableregion and yield amplification products which ideally provide enoughvariability to distinguish each individual bioagent, and which areamenable to molecular mass analysis. In some embodiments, the highlyconserved sequence regions exhibit between about 80-100%, or betweenabout 90-100%, or between about 95-100% identity, or between about99-100% identity. The molecular mass of a given amplification productprovides a means of identifying the bioagent from which it was obtained,due to the variability of the variable region. Thus design of theprimers requires selection of a variable region with appropriatevariability to resolve the identity of a given bioagent. Bioagentidentifying amplicons are ideally specific to the identity of thebioagent.

Properties of the primers may include any number of properties relatedto structure including, but not limited to: nucleobase length which maybe contiguous (linked together) or non-contiguous (for example, two ormore contiguous segments which are joined by a linker or loop moiety),modified or universal nucleobases (used for specific purposes such asfor example, increasing hybridization affinity, preventing non-templatedadenylation and modifying molecular mass) percent complementarity to agiven target sequences.

Properties of the primers also include functional features including,but not limited to, orientation of hybridization (forward or reverse)relative to a nucleic acid template. The coding or sense strand is thestrand to which the forward priming primer hybridizes (forward primingorientation) while the reverse priming primer hybridizes to thenon-coding or antisense strand (reverse priming orientation). Thefunctional properties of a given primer pair also include the generictemplate nucleic acid to which the primer pair hybridizes. For example,identification of bioagents can be accomplished at different levelsusing primers suited to resolution of each individual level ofidentification. Broad range survey primers are designed with theobjective of identifying a bioagent as a member of a particular division(e.g., an order, family, genus or other such grouping of bioagents abovethe species level of bioagents). In some embodiments, broad range surveyintelligent primers are capable of identification of bioagents at thespecies or sub-species level. Other primers may have the functionalityof producing bioagent identifying amplicons for members of a giventaxonomic genus, clade, species, sub-species or genotype (includinggenetic variants which may include presence of virulence genes orantibiotic resistance genes or mutations). Additional functionalproperties of primer pairs include the functionality of performingamplification either singly (single primer pair per amplificationreaction vessel) or in a multiplex fashion (multiple primer pairs andmultiple amplification reactions within a single reaction vessel).

As used herein, the terms “purified” or “substantially purified” referto molecules, either nucleic or amino acid sequences, that are removedfrom their natural environment, isolated or separated, and are at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated. An “isolatedpolynucleotide” or “isolated oligonucleotide” is therefore asubstantially purified polynucleotide.

The term “reverse transcriptase” refers to an enzyme having the abilityto transcribe DNA from an RNA template. This enzymatic activity is knownas reverse transcriptase activity. Reverse transcriptase activity isdesirable in order to obtain DNA from RNA viruses which can then beamplified and analyzed by the methods of the present invention.

The term “ribosomal RNA” or “rRNA” refers to the primary ribonucleicacid constituent of ribosomes. Ribosomes are the protein-manufacturingorganelles of cells and exist in the cytoplasm. Ribosomal RNAs aretranscribed from the DNA genes encoding them.

The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture (e.g., microbiological cultures). On the other hand, it is meantto include both biological and environmental samples. A sample mayinclude a specimen of synthetic origin. Biological samples may beanimal, including human, fluid, solid (e.g., stool) or tissue, as wellas liquid and solid food and feed products and ingredients such as dairyitems, vegetables, meat and meat by-products, and waste. Biologicalsamples may be obtained from all of the various families of domesticanimals, as well as feral or wild animals, including, but not limitedto, such animals as ungulates, bear, fish, lagamorphs, rodents, etc.Environmental samples include environmental material such as surfacematter, soil, water, air and industrial samples, as well as samplesobtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention. The term “source of target nucleic acid” refers toany sample that contains nucleic acids (RNA or DNA). Particularlypreferred sources of target nucleic acids are biological samplesincluding, but not limited to blood, saliva, cerebral spinal fluid,pleural fluid, milk, lymph, sputum and semen.

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template that may or may not bepresent in a sample. Background template is often a contaminant. It maybe the result of carryover, or it may be due to the presence of nucleicacid contaminants sought to be purified away from the sample. Forexample, nucleic acids from organisms other than those to be detectedmay be present as background in a test sample.

A “segment” is defined herein as a region of nucleic acid within atarget sequence.

The “self-sustained sequence replication reaction” (3SR) (Guatelli etal., Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum atProc. Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based invitro amplification system (Kwok et al., Proc. Natl. Acad. Sci.,86:1173-1177 [1989]) that can exponentially amplify RNA sequences at auniform temperature. The amplified RNA can then be utilized for mutationdetection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In thismethod, an oligonucleotide primer is used to add a phage RNA polymerasepromoter to the 5′ end of the sequence of interest. In a cocktail ofenzymes and substrates that includes a second primer, reversetranscriptase, RNase H, RNA polymerase and ribo- and deoxyribonucleosidetriphosphates, the target sequence undergoes repeated rounds oftranscription, cDNA synthesis and second-strand synthesis to amplify thearea of interest. The use of 3SR to detect mutations is kineticallylimited to screening small segments of DNA (e.g., 200-300 base pairs).

As used herein, the term ““sequence alignment”” refers to a listing ofmultiple DNA or amino acid sequences and aligns them to highlight theirsimilarities. The listings can be made using bioinformatics computerprograms.

In context of this invention, the term “speciating primer pair” refersto a primer pair designed to produce a bioagent identifying ampliconwith the diagnostic capability of identifying species members of a groupof genera or a particular genus of bioagents. Primer pair number 2249(SEQ ID NOs: 430:1321), for example, is a speciating primer pair used todistinguish Staphylococcus aureus from other species of the genusStaphylococcus.

As used herein, a “sub-species characteristic” is a geneticcharacteristic that provides the means to distinguish two members of thesame bioagent species. For example, one viral strain could bedistinguished from another viral strain of the same species bypossessing a genetic change (e.g., for example, a nucleotide deletion,addition or substitution) in one of the viral genes, such as theRNA-dependent RNA polymerase. Sub-species characteristics such asvirulence genes and drug-are responsible for the phenotypic differencesamong the different strains of bacteria.

As used herein, the term “target” is used in a broad sense to indicatethe gene or genomic region being amplified by the primers. Because thepresent invention provides a plurality of amplification products fromany given primer pair (depending on the bioagent being analyzed),multiple amplification products from different specific nucleic acidsequences may be obtained. Thus, the term “target” is not used to referto a single specific nucleic acid sequence. The “target” is sought to besorted out from other nucleic acid sequences and contains a sequencethat has at least partial complementarity with an oligonucleotideprimer. The target nucleic acid may comprise single- or double-strandedDNA or RNA. A “segment” is defined as a region of nucleic acid withinthe target sequence.

The term “template” refers to a strand of nucleic acid on which acomplementary copy is built from nucleoside triphosphates through theactivity of a template-dependent nucleic acid polymerase. Within aduplex the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theT_(m) of nucleic acids are well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (see e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985).Other references (e.g., Allawi, H. T. & SantaLucia, J., Jr.Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry36, 10581-94 (1997) include more sophisticated computations which takestructural and environmental, as well as sequence characteristics intoaccount for the calculation of T_(m).

The term “triangulation genotyping analysis” refers to a method ofgenotyping a bioagent by measurement of molecular masses or basecompositions of amplification products, corresponding to bioagentidentifying amplicons, obtained by amplification of regions of more thanone gene. In this sense, the term “triangulation” refers to a method ofestablishing the accuracy of information by comparing three or moretypes of independent points of view bearing on the same findings.Triangulation genotyping analysis carried out with a plurality oftriangulation genotyping analysis primers yields a plurality of basecompositions that then provide a pattern or “barcode” from which aspecies type can be assigned. The species type may represent apreviously known sub-species or strain, or may be a previously unknownstrain having a specific and previously unobserved base compositionbarcode indicating the existence of a previously unknown genotype.

As used herein, the term “triangulation genotyping analysis primer pair”is a primer pair designed to produce bioagent identifying amplicons fordetermining species types in a triangulation genotyping analysis.

The employment of more than one bioagent identifying amplicon foridentification of a bioagent is herein referred to as “triangulationidentification.” Triangulation identification is pursued by analyzing aplurality of bioagent identifying amplicons produced with differentprimer pairs. This process is used to reduce false negative and falsepositive signals, and enable reconstruction of the origin of hybrid orotherwise engineered bioagents. For example, identification of the threepart toxin genes typical of B. anthracis (Bowen et al., J. Appl.Microbiol., 1999, 87, 270-278) in the absence of the expected signaturesfrom the B. anthracis genome would suggest a genetic engineering event.

In the context of this invention, the term “unknown bioagent” may meaneither: (i) a bioagent whose existence is known (such as the well knownbacterial species Staphylococcus aureus for example) but which is notknown to be in a sample to be analyzed, or (ii) a bioagent whoseexistence is not known (for example, the SARS coronavirus was unknownprior to April 2003). For example, if the method for identification ofcoronaviruses disclosed in commonly owned U.S. patent Ser. No.10/829,826 (incorporated herein by reference in its entirety) was to beemployed prior to April 2003 to identify the SARS coronavirus in aclinical sample, both meanings of “unknown” bioagent are applicablesince the SARS coronavirus was unknown to science prior to April, 2003and since it was not known what bioagent (in this case a coronavirus)was present in the sample. On the other hand, if the method of U.S.patent Ser. No. 10/829,826 was to be employed subsequent to April 2003to identify the SARS coronavirus in a clinical sample, only the firstmeaning (i) of “unknown” bioagent would apply since the SARS coronavirusbecame known to science subsequent to April 2003 and since it was notknown what bioagent was present in the sample.

The term “variable sequence” as used herein refers to differences innucleic acid sequence between two nucleic acids. For example, the genesof two different bacterial species may vary in sequence by the presenceof single base substitutions and/or deletions or insertions of one ormore nucleotides. These two forms of the structural gene are said tovary in sequence from one another. In the context of the presentinvention, “viral nucleic acid” includes, but is not limited to, DNA,RNA, or DNA that has been obtained from viral RNA, such as, for example,by performing a reverse transcription reaction. Viral RNA can either besingle-stranded (of positive or negative polarity) or double-stranded.

The term “virus” refers to obligate, ultramicroscopic, parasites thatare incapable of autonomous replication (i.e., replication requires theuse of the host cell's machinery). Viruses can survive outside of a hostcell but cannot replicate.

The term “wild-type” refers to a gene or a gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified”, “mutant” or “polymorphic” refers to a gene or gene productthat displays modifications in sequence and or functional properties(i.e., altered characteristics) when compared to the wild-type gene orgene product. It is noted that naturally-occurring mutants can beisolated; these are identified by the fact that they have alteredcharacteristics when compared to the wild-type gene or gene product.

As used herein, a “wobble base” is a variation in a codon found at thethird nucleotide position of a DNA triplet. Variations in conservedregions of sequence are often found at the third nucleotide position dueto redundancy in the amino acid code.

DETAILED DESCRIPTION OF EMBODIMENTS

A. Bioagent Identifying Amplicons

The present invention provides methods for detection and identificationof unknown bioagents using bioagent identifying amplicons. Primers areselected to hybridize to conserved sequence regions of nucleic acidsderived from a bioagent, and which bracket variable sequence regions toyield a bioagent identifying amplicon, which can be amplified and whichis amenable to molecular mass determination. The molecular mass thenprovides a means to uniquely identify the bioagent without a requirementfor prior knowledge of the possible identity of the bioagent. Themolecular mass or corresponding base composition signature of theamplification product is then matched against a database of molecularmasses or base composition signatures. A match is obtained when anexperimentally-determined molecular mass or base composition of ananalyzed amplification product is compared with known molecular massesor base compositions of known bioagent identifying amplicons and theexperimentally determined molecular mass or base composition is the sameas the molecular mass or base composition of one of the known bioagentidentifying amplicons. Alternatively, the experimentally-determinedmolecular mass or base composition may be within experimental error ofthe molecular mass or base composition of a known bioagent identifyingamplicon and still be classified as a match. In some cases, the matchmay also be classified using a probability of match model such as themodels described in U.S. Ser. No. 11/073,362, which is commonly ownedand incorporated herein by reference in entirety. Furthermore, themethod can be applied to rapid parallel multiplex analyses, the resultsof which can be employed in a triangulation identification strategy. Thepresent method provides rapid throughput and does not require nucleicacid sequencing of the amplified target sequence for bioagent detectionand identification.

Despite enormous biological diversity, all forms of life on earth sharesets of essential, common features in their genomes. Since genetic dataprovide the underlying basis for identification of bioagents by themethods of the present invention, it is necessary to select segments ofnucleic acids which ideally provide enough variability to distinguisheach individual bioagent and whose molecular mass is amenable tomolecular mass determination.

Unlike bacterial genomes, which exhibit conservation of numerous genes(i.e. housekeeping genes) across all organisms, viruses do not share agene that is essential and conserved among all virus families.Therefore, viral identification is achieved within smaller groups ofrelated viruses, such as members of a particular virus family or genus.For example, RNA-dependent RNA polymerase is present in allsingle-stranded RNA viruses and can be used for broad priming as well asresolution within the virus family.

In some embodiments of the present invention, at least one bacterialnucleic acid segment is amplified in the process of identifying thebacterial bioagent. Thus, the nucleic acid segments that can beamplified by the primers disclosed herein and that provide enoughvariability to distinguish each individual bioagent and whose molecularmasses are amenable to molecular mass determination are herein describedas bioagent identifying amplicons.

In some embodiments of the present invention, bioagent identifyingamplicons comprise from about 45 to about 150 nucleobases (i.e. fromabout 45 to about 200 linked nucleosides), although both longer andshort regions may be used. One of ordinary skill in the art willappreciate that the invention embodies compounds of 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, and 150 nucleobases in length, or any rangetherewithin.

It is the combination of the portions of the bioagent nucleic acidsegment to which the primers hybridize (hybridization sites) and thevariable region between the primer hybridization sites that comprisesthe bioagent identifying amplicon. Thus, it can be said that a givenbioagent identifying amplicon is “defined by” a given pair of primers.

In some embodiments, bioagent identifying amplicons amenable tomolecular mass determination which are produced by the primers describedherein are either of a length, size or mass compatible with theparticular mode of molecular mass determination or compatible with ameans of providing a predictable fragmentation pattern in order toobtain predictable fragments of a length compatible with the particularmode of molecular mass determination. Such means of providing apredictable fragmentation pattern of an amplification product include,but are not limited to, cleavage with chemical reagents, restrictionenzymes or cleavage primers, for example. Thus, in some embodiments,bioagent identifying amplicons are larger than 150 nucleobases and areamenable to molecular mass determination following restrictiondigestion. Methods of using restriction enzymes and cleavage primers arewell known to those with ordinary skill in the art.

In some embodiments, amplification products corresponding to bioagentidentifying amplicons are obtained using the polymerase chain reaction(PCR) that is a routine method to those with ordinary skill in themolecular biology arts. Other amplification methods may be used such asligase chain reaction (LCR), low-stringency single primer PCR, andmultiple strand displacement amplification (MDA). These methods are alsoknown to those with ordinary skill.

B. Primers and Primer Pairs

In some embodiments, the primers are designed to bind to conservedsequence regions of a bioagent identifying amplicon that flank anintervening variable region and yield amplification products whichprovide variability sufficient to distinguish each individual bioagent,and which are amenable to molecular mass analysis. In some embodiments,the highly conserved sequence regions exhibit between about 80-100%, orbetween about 90-100%, or between about 95-100% identity, or betweenabout 99-100% identity. The molecular mass of a given amplificationproduct provides a means of identifying the bioagent from which it wasobtained, due to the variability of the variable region. Thus, design ofthe primers involves selection of a variable region with sufficientvariability to resolve the identity of a given bioagent. In someembodiments, bioagent identifying amplicons are specific to the identityof the bioagent.

In some embodiments, identification of bioagents is accomplished atdifferent levels using primers suited to resolution of each individuallevel of identification. Broad range survey primers are designed withthe objective of identifying a bioagent as a member of a particulardivision (e.g., an order, family, genus or other such grouping ofbioagents above the species level of bioagents). In some embodiments,broad range survey intelligent primers are capable of identification ofbioagents at the species or sub-species level. Examples of broad rangesurvey primers include, but are not limited to: primer pair numbers: 346(SEQ ID NOs: 202:1110), 347 (SEQ ID NOs: 560:1278), 348 SEQ ID NOs:706:895), and 361 (SEQ ID NOs: 697:1398) which target DNA encoding 16SrRNA, and primer pair numbers 349 (SEQ ID NOs: 401:1156) and 360 (SEQ IDNOs: 409:1434) which target DNA encoding 23S rRNA.

In some embodiments, drill-down primers are designed with the objectiveof identifying a bioagent at the sub-species level (including strains,subtypes, variants and isolates) based on sub-species characteristicswhich may, for example, include single nucleotide polymorphisms (SNPs),variable number tandem repeats (VNTRs), deletions, drug resistancemutations or any other modification of a nucleic acid sequence of abioagent relative to other members of a species having differentsub-species characteristics. Drill-down intelligent primers are notalways required for identification at the sub-species level becausebroad range survey intelligent primers may, in some cases providesufficient identification resolution to accomplishing thisidentification objective. Examples of drill-down primers include, butare not limited to: confirmation primer pairs such as primer pairnumbers 351 (SEQ ID NOs: 355:1423) and 353 (SEQ ID NOs: 220:1394), whichtarget the pX01 virulence plasmid of Bacillus anthracis. Other examplesof drill-down primer pairs are found in sets of triangulation genotypingprimer pairs such as, for example, the primer pair number 2146 (SEQ IDNOs: 437:1137) which targets the arcC gene (encoding carmabate kinase)and is included in an 8 primer pair panel or kit for use in genotypingStaphylococcus aureus, or in other panels or kits of primer pairs usedfor determining drug-resistant bacterial strains, such as, for example,primer pair number 2095 (SEQ ID NOs: 456:1261) which targets the pv-lukgene (encoding Panton-Valentine leukocidin) and is included in an 8primer pair panel or kit for use in identification of drug resistantstrains of Staphylococcus aureus.

A representative process flow diagram used for primer selection andvalidation process is outlined in FIG. 1. For each group of organisms,candidate target sequences are identified (200) from which nucleotidealignments are created (210) and analyzed (220). Primers are thendesigned by selecting appropriate priming regions (230) to facilitatethe selection of candidate primer pairs (240). The primer pairs are thensubjected to in silico analysis by electronic PCR (ePCR) (300) whereinbioagent identifying amplicons are obtained from sequence databases suchas GenBank or other sequence collections (310) and checked forspecificity in silico (320). Bioagent identifying amplicons obtainedfrom GenBank sequences (310) can also be analyzed by a probability modelwhich predicts the capability of a given amplicon to identify unknownbioagents such that the base compositions of amplicons with favorableprobability scores are then stored in a base composition database (325).Alternatively, base compositions of the bioagent identifying ampliconsobtained from the primers and GenBank sequences can be directly enteredinto the base composition database (330). Candidate primer pairs (240)are validated by testing their ability to hybridize to target nucleicacid by an in vitro amplification by a method such as PCR analysis (400)of nucleic acid from a collection of organisms (410). Amplificationproducts thus obtained are analyzed by gel electrophoresis or by massspectrometry to confirm the sensitivity, specificity and reproducibilityof the primers used to obtain the amplification products (420).

Many of the important pathogens, including the organisms of greatestconcern as biowarfare agents, have been completely sequenced. Thiseffort has greatly facilitated the design of primers for the detectionof unknown bioagents. The combination of broad-range priming withdivision-wide and drill-down priming has been used very successfully inseveral applications of the technology, including environmentalsurveillance for biowarfare threat agents and clinical sample analysisfor medically important pathogens.

Synthesis of primers is well known and routine in the art. The primersmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed.

In some embodiments primers are employed as compositions for use inmethods for identification of bacterial bioagents as follows: a primerpair composition is contacted with nucleic acid (such as, for example,bacterial DNA or DNA reverse transcribed from the rRNA) of an unknownbacterial bioagent. The nucleic acid is then amplified by a nucleic acidamplification technique, such as PCR for example, to obtain anamplification product that represents a bioagent identifying amplicon.The molecular mass of each strand of the double-stranded amplificationproduct is determined by a molecular mass measurement technique such asmass spectrometry for example, wherein the two strands of thedouble-stranded amplification product are separated during theionization process. In some embodiments, the mass spectrometry iselectrospray Fourier transform ion cyclotron resonance mass spectrometry(ESI-FTICR-MS) or electrospray time of flight mass spectrometry(ESI-TOF-MS). A list of possible base compositions can be generated forthe molecular mass value obtained for each strand and the choice of thecorrect base composition from the list is facilitated by matching thebase composition of one strand with a complementary base composition ofthe other strand. The molecular mass or base composition thus determinedis then compared with a database of molecular masses or basecompositions of analogous bioagent identifying amplicons for known viralbioagents. A match between the molecular mass or base composition of theamplification product and the molecular mass or base composition of ananalogous bioagent identifying amplicon for a known viral bioagentindicates the identity of the unknown bioagent. In some embodiments, theprimer pair used is one of the primer pairs of Table 2. In someembodiments, the method is repeated using one or more different primerpairs to resolve possible ambiguities in the identification process orto improve the confidence level for the identification assignment.

In some embodiments, a bioagent identifying amplicon may be producedusing only a single primer (either the forward or reverse primer of anygiven primer pair), provided an appropriate amplification method ischosen, such as, for example, low stringency single primer PCR(LSSP-PCR). Adaptation of this amplification method in order to producebioagent identifying amplicons can be accomplished by one with ordinaryskill in the art without undue experimentation.

In some embodiments, the oligonucleotide primers are broad range surveyprimers which hybridize to conserved regions of nucleic acid encodingthe hexon gene of all (or between 80% and 100%, between 85% and 100%,between 90% and 100% or between 95% and 100%) known bacteria and producebacterial bioagent identifying amplicons.

In some cases, the molecular mass or base composition of a bacterialbioagent identifying amplicon defined by a broad range survey primerpair does not provide enough resolution to unambiguously identify abacterial bioagent at or below the species level. These cases benefitfrom further analysis of one or more bacterial bioagent identifyingamplicons generated from at least one additional broad range surveyprimer pair or from at least one additional division-wide primer pair.The employment of more than one bioagent identifying amplicon foridentification of a bioagent is herein referred to as triangulationidentification.

In other embodiments, the oligonucleotide primers are division-wideprimers which hybridize to nucleic acid encoding genes of species withina genus of bacteria. In other embodiments, the oligonucleotide primersare drill-down primers which enable the identification of sub-speciescharacteristics. Drill down primers provide the functionality ofproducing bioagent identifying amplicons for drill-down analyses such asstrain typing when contacted with nucleic acid under amplificationconditions. Identification of such sub-species characteristics is oftencritical for determining proper clinical treatment of viral infections.In some embodiments, sub-species characteristics are identified usingonly broad range survey primers and division-wide and drill-down primersare not used.

In some embodiments, the primers used for amplification hybridize to andamplify genomic DNA, and DNA of bacterial plasmids.

In some embodiments, various computer software programs may be used toaid in design of primers for amplification reactions such as PrimerPremier 5 (Premier Biosoft, Palo Alto, Calif.) or OLIGO Primer AnalysisSoftware (Molecular Biology Insights, Cascade, Colo.). These programsallow the user to input desired hybridization conditions such as meltingtemperature of a primer-template duplex for example. In someembodiments, an in silico PCR search algorithm, such as (ePCR) is usedto analyze primer specificity across a plurality of template sequenceswhich can be readily obtained from public sequence databases such asGenBank for example. An existing RNA structure search algorithm (Mackeet al., Nucl. Acids Res., 2001, 29, 472-44735, which is incorporatedherein by reference in its entirety) has been modified to include PCRparameters such as hybridization conditions, mismatches, andthermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A.,1998, 95, 1460-1465, which is incorporated herein by reference in itsentirety). This also provides information on primer specificity of theselected primer pairs. In some embodiments, the hybridization conditionsapplied to the algorithm can limit the results of primer specificityobtained from the algorithm. In some embodiments, the meltingtemperature threshold for the primer template duplex is specified to be35° C. or a higher temperature. In some embodiments the number ofacceptable mismatches is specified to be seven mismatches or less. Insome embodiments, the buffer components and concentrations and primerconcentrations may be specified and incorporated into the algorithm, forexample, an appropriate primer concentration is about 250 nM andappropriate buffer components are 50 mM sodium or potassium and 1.5 mMMg²⁺.

One with ordinary skill in the art of design of amplification primerswill recognize that a given primer need not hybridize with 100%complementarity in order to effectively prime the synthesis of acomplementary nucleic acid strand in an amplification reaction.Moreover, a primer may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent. (e.g., for example, a loop structure or a hairpin structure). Theprimers of the present invention may comprise at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or at least99% sequence identity with any of the primers listed in Table 2. Thus,in some embodiments of the present invention, an extent of variation of70% to 100%, or any range therewithin, of the sequence identity ispossible relative to the specific primer sequences disclosed herein.Determination of sequence identity is described in the followingexample: a primer 20 nucleobases in length which is identical to another20 nucleobase primer having two non-identical residues has 18 of 20identical residues (18/20=0.9 or 90% sequence identity). In anotherexample, a primer 15 nucleobases in length having all residues identicalto a 15 nucleobase segment of primer 20 nucleobases in length would have15/20=0.75 or 75% sequence identity with the 20 nucleobase primer.

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for UNIX, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome embodiments, complementarity of primers with respect to theconserved priming regions of viral nucleic acid is between about 70% andabout 75% 80%. In other embodiments, homology, sequence identity orcomplementarity, is between about 75% and about 80%. In yet otherembodiments, homology, sequence identity or complementarity, is at least85%, at least 90%, at least 92%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or is 100%.

In some embodiments, the primers described herein comprise at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or100% (or any range therewithin) sequence identity with the primersequences specifically disclosed herein.

One with ordinary skill is able to calculate percent sequence identityor percent sequence homology and able to determine, without undueexperimentation, the effects of variation of primer sequence identity onthe function of the primer in its role in priming synthesis of acomplementary strand of nucleic acid for production of an amplificationproduct of a corresponding bioagent identifying amplicon.

In one embodiment, the primers are at least 13 nucleobases in length. Inanother embodiment, the primers are less than 36 nucleobases in length.

In some embodiments of the present invention, the oligonucleotideprimers are 13 to 35 nucleobases in length (13 to 35 linked nucleotideresidues). These embodiments comprise oligonucleotide primers 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34 or 35 nucleobases in length, or any range therewithin. Thepresent invention contemplates using both longer and shorter primers.Furthermore, the primers may also be linked to one or more other desiredmoieties, including, but not limited to, affinity groups, ligands,regions of nucleic acid that are not complementary to the nucleic acidto be amplified, labels, etc. Primers may also form hairpin structures.For example, hairpin primers may be used to amplify short target nucleicacid molecules. The presence of the hairpin may stabilize theamplification complex (see e.g., TAQMAN MicroRNA Assays, AppliedBiosystems, Foster City, Calif.).

In some embodiments, any oligonucleotide primer pair may have one orboth primers with less then 70% sequence homology with a correspondingmember of any of the primer pairs of Table 2 if the primer pair has thecapability of producing an amplification product corresponding to abioagent identifying amplicon. In other embodiments, any oligonucleotideprimer pair may have one or both primers with a length greater than 35nucleobases if the primer pair has the capability of producing anamplification product corresponding to a bioagent identifying amplicon.

In some embodiments, the function of a given primer may be substitutedby a combination of two or more primers segments that hybridize adjacentto each other or that are linked by a nucleic acid loop structure orlinker which allows a polymerase to extend the two or more primers in anamplification reaction.

In some embodiments, the primer pairs used for obtaining bioagentidentifying amplicons are the primer pairs of Table 2. In otherembodiments, other combinations of primer pairs are possible bycombining certain members of the forward primers with certain members ofthe reverse primers. An example can be seen in Table 2 for two primerpair combinations of forward primer 16S_EC_(—)789_(—)810_F (SEQ ID NO:206), with the reverse primers 16S_EC_(—)880_(—)894_R (SEQ ID NO: 796),or 16S_EC_(—)882_(—)899_R or (SEQ ID NO: 818). Arriving at a favorablealternate combination of primers in a primer pair depends upon theproperties of the primer pair, most notably the size of the bioagentidentifying amplicon that would be produced by the primer pair, whichpreferably is between about 45 to about 150 nucleobases in length.Alternatively, a bioagent identifying amplicon longer than 150nucleobases in length could be cleaved into smaller segments by cleavagereagents such as chemical reagents, or restriction enzymes, for example.

In some embodiments, the primers are configured to amplify nucleic acidof a bioagent to produce amplification products that can be measured bymass spectrometry and from whose molecular masses candidate basecompositions can be readily calculated.

In some embodiments, any given primer comprises a modificationcomprising the addition of a non-templated T residue to the 5′ end ofthe primer (i.e., the added T residue does not necessarily hybridize tothe nucleic acid being amplified). The addition of a non-templated Tresidue has an effect of minimizing the addition of non-templatedadenosine residues as a result of the non-specific enzyme activity ofTaq polymerase (Magnuson et al., Biotechniques, 1996, 21, 700-709), anoccurrence which may lead to ambiguous results arising from molecularmass analysis.

In some embodiments of the present invention, primers may contain one ormore universal bases. Because any variation (due to codon wobble in the3^(rd) position) in the conserved regions among species is likely tooccur in the third position of a DNA (or RNA) triplet, oligonucleotideprimers can be designed such that the nucleotide corresponding to thisposition is a base which can bind to more than one nucleotide, referredto herein as a “universal nucleobase.” For example, under this “wobble”pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C,and uridine (U) binds to U or C. Other examples of universal nucleobasesinclude nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes etal., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degeneratenucleotides dP or dK (Hill et al.), an acyclic nucleoside analogcontaining 5-nitroindazole (Van Aerschot et al., Nucleosides andNucleotides, 1995, 14, 1053-1056) or the purine analog1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al.,Nucl. Acids Res., 1996, 24, 3302-3306).

In some embodiments, to compensate for the somewhat weaker binding bythe wobble base, the oligonucleotide primers are designed such that thefirst and second positions of each triplet are occupied by nucleotideanalogs that bind with greater affinity than the unmodified nucleotide.Examples of these analogs include, but are not limited to,2,6-diaminopurine which binds to thymine, 5-propynyluracil (also knownas propynylated thymine) which binds to adenine and 5-propynylcytosineand phenoxazines, including G-clamp, which binds to G. Propynylatedpyrimidines are described in U.S. Pat. Nos. 5,645,985, 5,830,653 and5,484,908, each of which is commonly owned and incorporated herein byreference in its entirety. Propynylated primers are described in U.SPre-Grant Publication No. 2003-0170682, which is also commonly owned andincorporated herein by reference in its entirety. Phenoxazines aredescribed in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, each ofwhich is incorporated herein by reference in its entirety. G-clamps aredescribed in U.S. Pat. Nos. 6,007,992 and 6,028,183, each of which isincorporated herein by reference in its entirety.

In some embodiments, primer hybridization is enhanced using primerscontaining 5-propynyl deoxy-cytidine and deoxy-thymidine nucleotides.These modified primers offer increased affinity and base pairingselectivity.

In some embodiments, non-template primer tags are used to increase themelting temperature (T_(m)) of a primer-template duplex in order toimprove amplification efficiency. A non-template tag is at least threeconsecutive A or T nucleotide residues on a primer which are notcomplementary to the template. In any given non-template tag, A can bereplaced by C or G and T can also be replaced by C or G. AlthoughWatson-Crick hybridization is not expected to occur for a non-templatetag relative to the template, the extra hydrogen bond in a G-C pairrelative to an A-T pair confers increased stability of theprimer-template duplex and improves amplification efficiency forsubsequent cycles of amplification when the primers hybridize to strandssynthesized in previous cycles.

In other embodiments, propynylated tags may be used in a manner similarto that of the non-template tag, wherein two or more 5-propynylcytidineor 5-propynyluridine residues replace template matching residues on aprimer. In other embodiments, a primer contains a modifiedinternucleoside linkage such as a phosphorothioate linkage, for example.

In some embodiments, the primers contain mass-modifying tags. Reducingthe total number of possible base compositions of a nucleic acid ofspecific molecular weight provides a means of avoiding a persistentsource of ambiguity in determination of base composition ofamplification products. Addition of mass-modifying tags to certainnucleobases of a given primer will result in simplification of de novodetermination of base composition of a given bioagent identifyingamplicon from its molecular mass.

In some embodiments of the present invention, the mass modifiednucleobase comprises one or more of the following: for example,7-deaza-2′-deoxyadenosine-5-triphosphate,5-iodo-2′-deoxyuridine-5′-triphosphate,5-bromo-2′-deoxyuridine-5′-triphosphate,5-bromo-2′-deoxycytidine-5′-triphosphate,5-iodo-2′-deoxycytidine-5′-triphosphate,5-hydroxy-2′-deoxyuridine-5′-triphosphate,4-thiothymidine-5′-triphosphate, 5-aza-2′-deoxyuridine-5′-triphosphate,5-fluoro-2′-deoxyuridine-5′-triphosphate,O6-methyl-2′-deoxyguanosine-5′-triphosphate,N2-methyl-2′-deoxyguanosine-5′-triphosphate,8-oxo-2′-deoxyguanosine-5′-triphosphate orthiothymidine-5′-triphosphate. In some embodiments, the mass-modifiednucleobase comprises ¹⁵N or ¹³C or both ¹⁵N and ¹³C.

In some embodiments, multiplex amplification is performed where multiplebioagent identifying amplicons are amplified with a plurality of primerpairs. The advantages of multiplexing are that fewer reaction containers(for example, wells of a 96- or 384-well plate) are needed for eachmolecular mass measurement, providing time, resource and cost savingsbecause additional bioagent identification data can be obtained within asingle analysis. Multiplex amplification methods are well known to thosewith ordinary skill and can be developed without undue experimentation.However, in some embodiments, one useful and non-obvious step inselecting a plurality candidate bioagent identifying amplicons formultiplex amplification is to ensure that each strand of eachamplification product will be sufficiently different in molecular massthat mass spectral signals will not overlap and lead to ambiguousanalysis results. In some embodiments, a 10 Da difference in mass of twostrands of one or more amplification products is sufficient to avoidoverlap of mass spectral peaks.

In some embodiments, as an alternative to multiplex amplification,single amplification reactions can be pooled before analysis by massspectrometry. In these embodiments, as for multiplex amplificationembodiments, it is useful to select a plurality of candidate bioagentidentifying amplicons to ensure that each strand of each amplificationproduct will be sufficiently different in molecular mass that massspectral signals will not overlap and lead to ambiguous analysisresults.

C Determination of Molecular Mass of Bioagent Identifying Amplicons

In some embodiments, the molecular mass of a given bioagent identifyingamplicon is determined by mass spectrometry. Mass spectrometry hasseveral advantages, not the least of which is high bandwidthcharacterized by the ability to separate (and isolate) many molecularpeaks across a broad range of mass to charge ratio (m/z). Thus massspectrometry is intrinsically a parallel detection scheme without theneed for radioactive or fluorescent labels, since every amplificationproduct is identified by its molecular mass. The current state of theart in mass spectrometry is such that less than femtomole quantities ofmaterial can be readily analyzed to afford information about themolecular contents of the sample. An accurate assessment of themolecular mass of the material can be quickly obtained, irrespective ofwhether the molecular weight of the sample is several hundred, or inexcess of one hundred thousand atomic mass units (amu) or Daltons.

In some embodiments, intact molecular ions are generated fromamplification products using one of a variety of ionization techniquesto convert the sample to gas phase. These ionization methods include,but are not limited to, electrospray ionization (ES), matrix-assistedlaser desorption ionization (MALDI) and fast atom bombardment (FAB).Upon ionization, several peaks are observed from one sample due to theformation of ions with different charges. Averaging the multiplereadings of molecular mass obtained from a single mass spectrum affordsan estimate of molecular mass of the bioagent identifying amplicon.Electrospray ionization mass spectrometry (ESI-MS) is particularlyuseful for very high molecular weight polymers such as proteins andnucleic acids having molecular weights greater than 10 kDa, since ityields a distribution of multiply-charged molecules of the samplewithout causing a significant amount of fragmentation.

The mass detectors used in the methods of the present invention include,but are not limited to, Fourier transform ion cyclotron resonance massspectrometry (FT-ICR-MS), time of flight (TOF), ion trap, quadrupole,magnetic sector, Q-TOF, and triple quadrupole.

D. Base Compositions of Bioagent Identifying Amplicons

Although the molecular mass of amplification products obtained usingintelligent primers provides a means for identification of bioagents,conversion of molecular mass data to a base composition signature isuseful for certain analyses. As used herein, “base composition” is theexact number of each nucleobase (A, T, C and G) determined from themolecular mass of a bioagent identifying amplicon. In some embodiments,a base composition provides an index of a specific organism. Basecompositions can be calculated from known sequences of known bioagentidentifying amplicons and can be experimentally determined by measuringthe molecular mass of a given bioagent identifying amplicon, followed bydetermination of all possible base compositions which are consistentwith the measured molecular mass within acceptable experimental error.The following example illustrates determination of base composition froman experimentally obtained molecular mass of a 46-mer amplificationproduct originating at position 1337 of the 16S rRNA of Bacillusanthracis. The forward and reverse strands of the amplification producthave measured molecular masses of 14208 and 14079 Da, respectively. Thepossible base compositions derived from the molecular masses of theforward and reverse strands for the B. anthracis products are listed inTable 1. TABLE 1 Possible Base Compositions for B. anthracis 46merAmplification Product Calc. Mass Mass Error Base Calc. Mass Mass ErrorBase Forward Forward Composition of Reverse Reverse Composition ofStrand Strand Forward Strand Strand Strand Reverse Strand 14208.29350.079520 A1 G17 C10 T18 14079.2624 0.080600 A0 G14 C13 T19 14208.31600.056980 A1 G20 C15 T10 14079.2849 0.058060 A0 G17 C18 T11 14208.33860.034440 A1 G23 C20 T2 14079.3075 0.035520 A0 G20 C23 T3 14208.30740.065560 A6 G11 C3 T26 14079.2538 0.089180 A5 G5 C1 T35 14208.33000.043020 A6 G14 C8 T18 14079.2764 0.066640 A5 G8 C6 T27 14208.35250.020480 A6 G17 C13 T10 14079.2989 0.044100 A5 G11 C11 T19 14208.37510.002060 A6 G20 C18 T2 14079.3214 0.021560 A5 G14 C16 T11 14208.34390.029060 A11 G8 C1 T26 14079.3440 0.000980 A5 G17 C21 T3 14208.36650.006520 A11 G11 C6 T18 14079.3129 0.030140 A10 G5 C4 T27 14208.38900.016020 A11 G14 C11 T10 14079.3354 0.007600 A10 G8 C9 T19 14208.41160.038560 A11 G17 C16 T2 14079.3579 0.014940 A10 G11 C14 T11 14208.40300.029980 A16 G8 C4 T18 14079.3805 0.037480 A10 G14 C19 T3 14208.42550.052520 A16 G11 C9 T10 14079.3494 0.006360 A15 G2 C2 T27 14208.44810.075060 A16 G14 C14 T2 14079.3719 0.028900 A15 G5 C7 T19 14208.43950.066480 A21 G5 C2 T18 14079.3944 0.051440 A15 G8 C12 T11 14208.46200.089020 A21 G8 C7 T10 14079.4170 0.073980 A15 G11 C17 T3 — — —14079.4084 0.065400 A20 G2 C5 T19 — — — 14079.4309 0.087940 A20 G5 C10T13

Among the 16 possible base compositions for the forward strand and the18 possible base compositions for the reverse strand that werecalculated, only one pair (shown in bold) are complementary basecompositions, which indicates the true base composition of theamplification product. It should be recognized that this logic isapplicable for determination of base compositions of any bioagentidentifying amplicon, regardless of the class of bioagent from which thecorresponding amplification product was obtained.

In some embodiments, assignment of previously unobserved basecompositions (also known as “true unknown base compositions”) to a givenphylogeny can be accomplished via the use of pattern classifier modelalgorithms. Base compositions, like sequences, vary slightly from strainto strain within species, for example. In some embodiments, the patternclassifier model is the mutational probability model. On otherembodiments, the pattern classifier is the polytope model. Themutational probability model and polytope model are both commonly ownedand described in U.S. patent application Ser. No. 11/073,362 which isincorporated herein by reference in entirety.

In one embodiment, it is possible to manage this diversity by building“base composition probability clouds” around the composition constraintsfor each species. This permits identification of organisms in a fashionsimilar to sequence analysis. A “pseudo four-dimensional plot” can beused to visualize the concept of base composition probability clouds.Optimal primer design requires optimal choice of bioagent identifyingamplicons and maximizes the separation between the base compositionsignatures of individual bioagents. Areas where clouds overlap indicateregions that may result in a misclassification, a problem which isovercome by a triangulation identification process using bioagentidentifying amplicons not affected by overlap of base compositionprobability clouds.

In some embodiments, base composition probability clouds provide themeans for screening potential primer pairs in order to avoid potentialmisclassifications of base compositions. In other embodiments, basecomposition probability clouds provide the means for predicting theidentity of a bioagent whose assigned base composition was notpreviously observed and/or indexed in a bioagent identifying ampliconbase composition database due to evolutionary transitions in its nucleicacid sequence. Thus, in contrast to probe-based techniques, massspectrometry determination of base composition does not require priorknowledge of the composition or sequence in order to make themeasurement.

The present invention provides bioagent classifying information similarto DNA sequencing and phylogenetic analysis at a level sufficient toidentify a given bioagent. Furthermore, the process of determination ofa previously unknown base composition for a given bioagent (for example,in a case where sequence information is unavailable) has downstreamutility by providing additional bioagent indexing information with whichto populate base composition databases. The process of future bioagentidentification is thus greatly improved as more BCS indexes becomeavailable in base composition databases.

E. Triangulation Identification

In some cases, a molecular mass of a single bioagent identifyingamplicon alone does not provide enough resolution to unambiguouslyidentify a given bioagent. The employment of more than one bioagentidentifying amplicon for identification of a bioagent is herein referredto as “triangulation identification.” Triangulation identification ispursued by determining the molecular masses of a plurality of bioagentidentifying amplicons selected within a plurality of housekeeping genes.This process is used to reduce false negative and false positivesignals, and enable reconstruction of the origin of hybrid or otherwiseengineered bioagents. For example, identification of the three parttoxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol.,1999, 87, 270-278) in the absence of the expected signatures from the B.anthracis genome would suggest a genetic engineering event.

In some embodiments, the triangulation identification process can bepursued by characterization of bioagent identifying amplicons in amassively parallel fashion using the polymerase chain reaction (PCR),such as multiplex PCR where multiple primers are employed in the sameamplification reaction mixture, or PCR in multi-well plate formatwherein a different and unique pair of primers is used in multiple wellscontaining otherwise identical reaction mixtures. Such multiplex andmulti-well PCR methods are well known to those with ordinary skill inthe arts of rapid throughput amplification of nucleic acids. In otherrelated embodiments, one PCR reaction per well or container may becarried out, followed by an amplicon pooling step wherein theamplification products of different wells are combined in a single wellor container which is then subjected to molecular mass analysis. Thecombination of pooled amplicons can be chosen such that the expectedranges of molecular masses of individual amplicons are not overlappingand thus will not complicate identification of signals.

F. Codon Base Composition Analysis

In some embodiments of the present invention, one or more nucleotidesubstitutions within a codon of a gene of an infectious organism conferdrug resistance upon an organism which can be determined by codon basecomposition analysis. The organism can be a bacterium, virus, fungus orprotozoan.

In some embodiments, the amplification product containing the codonbeing analyzed is of a length of about 35 to about 200 nucleobases. Theprimers employed in obtaining the amplification product can hybridize toupstream and downstream sequences directly adjacent to the codon, or canhybridize to upstream and downstream sequences one or more sequencepositions away from the codon. The primers may have between about 70% to100% sequence complementarity with the sequence of the gene containingthe codon being analyzed.

In some embodiments, the codon base composition analysis is undertaken

In some embodiments, the codon analysis is undertaken for the purpose ofinvestigating genetic disease in an individual. In other embodiments,the codon analysis is undertaken for the purpose of investigating a drugresistance mutation or any other deleterious mutation in an infectiousorganism such as a bacterium, virus, fungus or protozoan. In someembodiments, the bioagent is a bacterium identified in a biologicalproduct.

In some embodiments, the molecular mass of an amplification productcontaining the codon being analyzed is measured by mass spectrometry.The mass spectrometry can be either electrospray (ESI) mass spectrometryor matrix-assisted laser desorption ionization (MALDI) massspectrometry. Time-of-flight (TOF) is an example of one mode of massspectrometry compatible with the analyses of the present invention.

The methods of the present invention can also be employed to determinethe relative abundance of drug resistant strains of the organism beinganalyzed. Relative abundances can be calculated from amplitudes of massspectral signals with relation to internal calibrants. In someembodiments, known quantities of internal amplification calibrants canbe included in the amplification reactions and abundances of analyteamplification product estimated in relation to the known quantities ofthe calibrants.

In some embodiments, upon identification of one or more drug-resistantstrains of an infectious organism infecting an individual, one or morealternative treatments can be devised to treat the individual.

G. Determination of the Quantity of a Bioagent

In some embodiments, the identity and quantity of an unknown bioagentcan be determined using the process illustrated in FIG. 2. Primers (500)and a known quantity of a calibration polynucleotide (505) are added toa sample containing nucleic acid of an unknown bioagent. The totalnucleic acid in the sample is then subjected to an amplificationreaction (510) to obtain amplification products. The molecular masses ofamplification products are determined (515) from which are obtainedmolecular mass and abundance data. The molecular mass of the bioagentidentifying amplicon (520) provides the means for its identification(525) and the molecular mass of the calibration amplicon obtained fromthe calibration polynucleotide (530) provides the means for itsidentification (535). The abundance data of the bioagent identifyingamplicon is recorded (540) and the abundance data for the calibrationdata is recorded (545), both of which are used in a calculation (550)which determines the quantity of unknown bioagent in the sample.

A sample comprising an unknown bioagent is contacted with a pair ofprimers that provide the means for amplification of nucleic acid fromthe bioagent, and a known quantity of a polynucleotide that comprises acalibration sequence. The nucleic acids of the bioagent and of thecalibration sequence are amplified and the rate of amplification isreasonably assumed to be similar for the nucleic acid of the bioagentand of the calibration sequence. The amplification reaction thenproduces two amplification products: a bioagent identifying amplicon anda calibration amplicon. The bioagent identifying amplicon and thecalibration amplicon should be distinguishable by molecular mass whilebeing amplified at essentially the same rate. Effecting differentialmolecular masses can be accomplished by choosing as a calibrationsequence, a representative bioagent identifying amplicon (from aspecific species of bioagent) and performing, for example, a 2-8nucleobase deletion or insertion within the variable region between thetwo priming sites. The amplified sample containing the bioagentidentifying amplicon and the calibration amplicon is then subjected tomolecular mass analysis by mass spectrometry, for example. The resultingmolecular mass analysis of the nucleic acid of the bioagent and of thecalibration sequence provides molecular mass data and abundance data forthe nucleic acid of the bioagent and of the calibration sequence. Themolecular mass data obtained for the nucleic acid of the bioagentenables identification of the unknown bioagent and the abundance dataenables calculation of the quantity of the bioagent, based on theknowledge of the quantity of calibration polynucleotide contacted withthe sample.

In some embodiments, construction of a standard curve where the amountof calibration polynucleotide spiked into the sample is varied providesadditional resolution and improved confidence for the determination ofthe quantity of bioagent in the sample. The use of standard curves foranalytical determination of molecular quantities is well known to onewith ordinary skill and can be performed without undue experimentation.

In some embodiments, multiplex amplification is performed where multiplebioagent identifying amplicons are amplified with multiple primer pairswhich also amplify the corresponding standard calibration sequences. Inthis or other embodiments, the standard calibration sequences areoptionally included within a single vector which functions as thecalibration polynucleotide. Multiplex amplification methods are wellknown to those with ordinary skill and can be performed without undueexperimentation.

In some embodiments, the calibrant polynucleotide is used as an internalpositive control to confirm that amplification conditions and subsequentanalysis steps are successful in producing a measurable amplicon. Evenin the absence of copies of the genome of a bioagent, the calibrationpolynucleotide should give rise to a calibration amplicon. Failure toproduce a measurable calibration amplicon indicates a failure ofamplification or subsequent analysis step such as amplicon purificationor molecular mass determination. Reaching a conclusion that suchfailures have occurred is in itself, a useful event.

In some embodiments, the calibration sequence is comprised of DNA. Insome embodiments, the calibration sequence is comprised of RNA.

In some embodiments, the calibration sequence is inserted into a vectorthat itself functions as the calibration polynucleotide. In someembodiments, more than one calibration sequence is inserted into thevector that functions as the calibration polynucleotide. Such acalibration polynucleotide is herein termed a “combination calibrationpolynucleotide.” The process of inserting polynucleotides into vectorsis routine to those skilled in the art and can be accomplished withoutundue experimentation. Thus, it should be recognized that thecalibration method should not be limited to the embodiments describedherein. The calibration method can be applied for determination of thequantity of any bioagent identifying amplicon when an appropriatestandard calibrant polynucleotide sequence is designed and used. Theprocess of choosing an appropriate vector for insertion of a calibrantis also a routine operation that can be accomplished by one withordinary skill without undue experimentation.

H. Identification of Bacteria

In other embodiments of the present invention, the primer pairs producebioagent identifying amplicons within stable and highly conservedregions of bacteria. The advantage to characterization of an amplicondefined by priming regions that fall within a highly conserved region isthat there is a low probability that the region will evolve past thepoint of primer recognition, in which case, the primer hybridization ofthe amplification step would fail. Such a primer set is thus useful as abroad range survey-type primer. In another embodiment of the presentinvention, the intelligent primers produce bioagent identifyingamplicons including a region which evolves more quickly than the stableregion described above. The advantage of characterization bioagentidentifying amplicon corresponding to an evolving genomic region is thatit is useful for distinguishing emerging strain variants or the presenceof virulence genes, drug resistance genes, or codon mutations thatinduce drug resistance.

The present invention also has significant advantages as a platform foridentification of diseases caused by emerging bacterial strains such as,for example, drug-resistant strains of Staphylococcus aureus. Thepresent invention eliminates the need for prior knowledge of bioagentsequence to generate hybridization probes. This is possible because themethods are not confounded by naturally occurring evolutionaryvariations occurring in the sequence acting as the template forproduction of the bioagent identifying amplicon. Measurement ofmolecular mass and determination of base composition is accomplished inan unbiased manner without sequence prejudice.

Another embodiment of the present invention also provides a means oftracking the spread of a bacterium, such as a particular drug-resistantstrain when a plurality of samples obtained from different locations areanalyzed by the methods described above in an epidemiological setting.In one embodiment, a plurality of samples from a plurality of differentlocations is analyzed with primer pairs which produce bioagentidentifying amplicons, a subset of which contains a specificdrug-resistant bacterial strain. The corresponding locations of themembers of the drug-resistant strain subset indicate the spread of thespecific drug-resistant strain to the corresponding locations.

I. Kits

The present invention also provides kits for carrying out the methodsdescribed herein. In some embodiments, the kit may comprise a sufficientquantity of one or more primer pairs to perform an amplificationreaction on a target polynucleotide from a bioagent to form a bioagentidentifying amplicon. In some embodiments, the kit may comprise from oneto fifty primer pairs, from one to twenty primer pairs, from one to tenprimer pairs, or from two to five primer pairs. In some embodiments, thekit may comprise one or more primer pairs recited in Table 2.

In some embodiments, the kit comprises one or more broad range surveyprimer(s), division wide primer(s), or drill-down primer(s), or anycombination thereof. If a given problem involves identification of aspecific bioagent, the solution to the problem may require the selectionof a particular combination of primers to provide the solution to theproblem. A kit may be designed so as to comprise particular primer pairsfor identification of a particular bioagent. A drill-down kit may beused, for example, to distinguish different genotypes or strains,drug-resistant, or otherwise. In some embodiments, the primer paircomponents of any of these kits may be additionally combined to compriseadditional combinations of broad range survey primers and division-wideprimers so as to be able to identify a bacterium.

In some embodiments, the kit contains standardized calibrationpolynucleotides for use as internal amplification calibrants. Internalcalibrants are described in commonly owned U.S. Patent Application Ser.No. 60/545,425 which is incorporated herein by reference in itsentirety.

In some embodiments, the kit comprises a sufficient quantity of reversetranscriptase (if RNA is to be analyzed for example), a DNA polymerase,suitable nucleoside triphosphates (including alternative dNTPs such asinosine or modified dNTPs such as the 5-propynyl pyrimidines or any dNTPcontaining molecular mass-modifying tags such as those described above),a DNA ligase, and/or reaction buffer, or any combination thereof, forthe amplification processes described above. A kit may further includeinstructions pertinent for the particular embodiment of the kit, suchinstructions describing the primer pairs and amplification conditionsfor operation of the method. A kit may also comprise amplificationreaction containers such as microcentrifuge tubes and the like. A kitmay also comprise reagents or other materials for isolating bioagentnucleic acid or bioagent identifying amplicons from amplification,including, for example, detergents, solvents, or ion exchange resinswhich may be linked to magnetic beads. A kit may also comprise a tableof measured or calculated molecular masses and/or base compositions ofbioagents using the primer pairs of the kit.

Some embodiments are kits that contain one or more survey bacterialprimer pairs represented by primer pair compositions wherein each memberof each pair of primers has 70% to 100% sequence identity with thecorresponding member from the group of primer pairs represented by anyof the primer pairs of Table 5. The survey primer pairs may includebroad range primer pairs which hybridize to ribosomal RNA, and may alsoinclude division-wide primer pairs which hybridize to housekeeping genessuch as rplB, tufB, rpoB, rpoC, valS, and infB, for example.

In some embodiments, a kit may contain one or more survey bacterialprimer pairs and one or more triangulation genotyping analysis primerpairs such as the primer pairs of Tables 8, 12, 14, 19, 21, 23, or 24.In some embodiments, the kit may represent a less expansive genotypinganalysis but include triangulation genotyping analysis primer pairs formore than one genus or species of bacteria. For example, a kit forsurveying nosocomial infections at a health care facility may include,for example, one or more broad range survey primer pairs, one or moredivision wide primer pairs, one or more Acinetobacter baumanniitriangulation genotyping analysis primer pairs and one or moreStaphylococcus aureus triangulation genotyping analysis primer pairs.One with ordinary skill will be capable of analyzing in silicoamplification data to determine which primer pairs will be able toprovide optimal identification resolution for the bacterial bioagents ofinterest.

In some embodiments, a kit may be assembled for identification ofstrains of bacteria involved in contamination of food. An example ofsuch a kit embodiment is a kit comprising one or more bacterial surveyprimer pairs of Table 5 with one or more triangulation genotypinganalysis primer pairs of Table 12 which provide strain resolvingcapabilities for identification of specific strains of Campylobacterjejuni.

Some embodiments of the kits are 96-well or 384-well plates with aplurality of wells containing any or all of the following components:dNTPs, buffer salts, Mg²⁺, betaine, and primer pairs. In someembodiments, a polymerase is also included in the plurality of wells ofthe 96-well or 384-well plates.

Some embodiments of the kit contain instructions for PCR and massspectrometry analysis of amplification products obtained using theprimer pairs of the kits.

Some embodiments of the kit include a barcode which uniquely identifiesthe kit and the components contained therein according to productionlots and may also include any other information relative to thecomponents such as concentrations, storage temperatures, etc. Thebarcode may also include analysis information to be read by opticalbarcode readers and sent to a computer controlling amplification,purification and mass spectrometric measurements. In some embodiments,the barcode provides access to a subset of base compositions in a basecomposition database which is in digital communication with basecomposition analysis software such that a base composition measured withprimer pairs from a given kit can be compared with known basecompositions of bioagent identifying amplicons defined by the primerpairs of that kit.

In some embodiments, the kit contains a database of base compositions ofbioagent identifying amplicons defined by the primer pairs of the kit.The database is stored on a convenient computer readable medium such asa compact disk or USB drive, for example.

In some embodiments, the kit includes a computer program stored on acomputer formatted medium (such as a compact disk or portable USB diskdrive, for example) comprising instructions which direct a processor toanalyze data obtained from the use of the primer pairs of the presentinvention. The instructions of the software transform data related toamplification products into a molecular mass or base composition whichis a useful concrete and tangible result used in identification and/orclassification of bioagents. In some embodiments, the kits of thepresent invention contain all of the reagents sufficient to carry outone or more of the methods described herein.

While the present invention has been described with specificity inaccordance with certain of its embodiments, the following examples serveonly to illustrate the invention and are not intended to limit the same.In order that the invention disclosed herein may be more efficientlyunderstood, examples are provided below. It should be understood thatthese examples are for illustrative purposes only and are not to beconstrued as limiting the invention in any manner.

EXAMPLES Example 1 Design and Validation of Primers that Define BioagentIdentifying Amplicons for Identification of Bacteria

For design of primers that define bacterial bioagent identifyingamplicons, a series of bacterial genome segment sequences were obtained,aligned and scanned for regions where pairs of PCR primers would amplifyproducts of about 45 to about 150 nucleotides in length and distinguishsubgroups and/or individual strains from each other by their molecularmasses or base compositions. A typical process shown in FIG. 1 isemployed for this type of analysis.

A database of expected base compositions for each primer region wasgenerated using an in silico PCR search algorithm, such as (ePCR). Anexisting RNA structure search algorithm (Macke et al., Nucl. Acids Res.,2001, 29, 4724-4735, which is incorporated herein by reference in itsentirety) has been modified to include PCR parameters such ashybridization conditions, mismatches, and thermodynamic calculations(SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, whichis incorporated herein by reference in its entirety). This also providesinformation on primer specificity of the selected primer pairs.

Table 2 represents a collection of primers (sorted by primer pairnumber) designed to identify bacteria using the methods describedherein. The primer pair number is an in-house database index number.Primer sites were identified on segments of genes, such as, for example,the 16S rRNA gene. The forward or reverse primer name shown in Table 2indicates the gene region of the bacterial genome to which the primerhybridizes relative to a reference sequence. In Table 2, for example,the forward primer name 16S_EC_(—)1077_(—)1106_F indicates that theforward primer (_F) hybridizes to residues 1077-1106 of the referencesequence represented by a sequence extraction of coordinates 4033120 . .. 4034661 from GenBank gi number 16127994 (as indicated in Table 3). Asan additional example: the forward primer nameBONTA_X52066_(—)450_(—)473 indicates that the primer hybridizes toresidues 450-437 of the gene encoding Clostridium botulinum neurotoxintype A (BoNT/A) represented by GenBank Accession No. X52066 (primer pairname codes appearing in Table 2 are defined in Table 3. One withordinary skill knows how to obtain individual gene sequences or portionsthereof from genomic sequences present in GenBank. In Table 2,Tp=5-propynyluracil; Cp=5-propynylcytosine; *=phosphorothioate linkage;I=inosine. T. GenBank Accession Numbers for reference sequences ofbacteria are shown in Table 3 (below). In some cases, the referencesequences are extractions from bacterial genomic sequences orcomplements thereof. TABLE 2 Primer Pairs for Identification of BacteriaPrimer +HC,86 Pair Forward SEQ Reverse SEQ Number Forward Primer NameForward Sequence ID NO: Reverse Primer Name Reverse Sequence ID NO: 116S_EC_1077_1106_F GTGAGATGTTGGGTTAAGTCCCGTAA 134 16S_EC_1175_1195_RGACGTCATCCCCACCTTCCTC 809 CGAG 2 16S_EC_1082_1106_FATGTTGGGTTAAGTCCCGCAACGAG 38 16S_EC_1175_1197_R TTGACGTCATCCCCACCTTCCTC1398 3 16S_EC_1090_1111_F TTAAGTCCCGCAACGATCGCAA 651 16S_EC_1175_1196_RTGACGTCATCCCCACCTTCCTC 1159 4 16S_EC_1222_1241_F GCTACACACGTGCTACAATG114 16S_EC_1303_1323_R CGAGTTGCAGACTGCGATCCG 787 5 16S_EC_1332_1353_FAAGTCGGAATCGCTAGTAATCG 10 16S_EC_1389_1407_R GACGGGCGGTGTGTACAAG 806 616S_EC_30_54_F TGAACGCTGGTGGCATGCTTAACAC 429 16S_EC_105_126_RTACGCATTACTCACCCGTCCGC 897 7 16S_EC_38_64_F GTGGCATGCCTAATACATGCAAGTCG136 16S_EC_101_120_R TTACTCACCCGTCCGCCGCT 1365 8 16S_EC_49_68_FTAACACATGCAAGTCGAACG 152 16S_EC_104_120_R TTACTCACCCGTCCGCC 1364 916S_EC_683_700_F GTGTAGCGGTGAAATGCG 137 16S_EC_774_795_RGTATCTAATCCTGTTTGCTCCC 839 10 16S_EC_713_732_F AGAACACCGATGGCGAAGGC 2116S_EC_789_809_R CGTGGACTACCAGGGTATCTA 798 11 16S_EC_785_806_FGGATTAGAGACCCTGGTAGTCC 118 16S_EC_880_897_R GGCCGTACTCCCCAGGCG 830 1216S_EC_785_810_F GGATTAGATACCCTGGTAGTCCACGC 119 16S_EC_80_897_2_RGGCCGTACTCCCCAGGCG 830 13 16S_EC_789_810_F TAGATACCCTGGTAGTCCACGC 20616S_EC_880_894_R CGTACTCCCCAGGCG 796 14 16S_EC_960_981_FTTCGATGCAACGCGAAGAACCT 672 16S_EC_1054_1073_R ACGAGCTGACGACAGCCATG 73515 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078_RACGACACGAGCTGACGAC 734 16 23S_EC_1826_1843_F CTGACACCTGCCCGGTGC 8023S_EC_1906_1924_R GACCGTTATAGTTACGGCC 805 17 23S_EC_2645_2669_FTCTGTCCCTAGTACGAGAGGACCGG 408 235_EC_2744_2761_R TGCTTAGATGCTTTCAGC 125218 23S_EC_2645_2669_2_F CTGTCCCTAGTACGAGAGGACCGG 83 23S_EC_2751_2767_RGTTTCATGCTTAGATGCTTTCAGC 846 19 23S_EC_493_518_FGGGGAGTGAAAGAGATCCTGAAACCG 125 23S_EC_551_571_R ACAAAAGGTACGCCGTCACCC717 20 23S_EC_493_518_2_F GGGCAGTGAAAGAGATCCTGAAACCG 12523S_EC_551_571_2_R ACAAAAGGCACGCCATCACCC 716 21 23S_EC_971_992_FCGAGAGGGAAACAACCCAGACC 66 23S_EC_1059_1077_R TGGCTGCTTCTAAGCCAAC 1282 22CAPC_BA_104_131_F GTTATTTAGCACTCGTTTTTAATCAG 139 CAPC_BA_180_205_RTGAATCTTGAAACACCATACGTAACG 1150 CC 23 CAPC_BA_114_133_FACTCGTTTTTAATCAGCCCG 20 CAPC_BA_185_205_R TGAATCTTGAAACACCATACG 1149 24CAPC_BA_274_303_F GATTATTGTTATCCTGTTATGCCATT 109 CAPC_BA_349_376_RGTAACCCTTGTCTTTGAATTGTATTTGC 837 TGAG 25 CAPC_BA_276_296_FTTATTGTTATCCTGTTATGCC 663 CAPC_BA_358_377_R GGTAACCCTTGTCTTTGAAT 834 26CAPC_BA_281_301_F GTTATCCTGTTATGCCATTTG 138 CAPC_BA_361_378_RTGGTAACCCTTGTCTTTG 1298 27 CAPC_BA_315_334_F CCGTGGTATTGGAGTTATTG 59CAPC_BA_361_378_R TGGTAACCCTTGTCTTTG 1298 28 CYA_BA_1055_1072_FGAAAGAGTTCGGATTGGG 92 CYA_BA_1112_1130_R TGTTGACCATGCTTCTTAG 1352 29CYA_BA_1349_1370_F ACAACGAGTACAATACAAGAC 12 CYA_BA_1447_1426_RCTTCTACATTTTTAGCCATCAC 800 30 CYA_BA_1353_1379_FCGAAGTACAATACAAGACAAAAGAAG 64 CYA_BA_1448_1467_R TGTTAACGGCTTCAAGACCC1342 G 31 CYA_BA_1359_1379_F ACAATACAAGACAAAAGAAGG 13 CYA_BA_1447_1461_RCGGCTTCAAGACCCC 794 32 CYA_BA_914_937_F CAGGTTTAGTACCAGAACATGCAG 53CYA_BA_999_1026_R ACCACTTTTAATAAGGTTTGTAGCTAAC 728 33 CYA_BA_916_935_FGGTTTAGTACCAGAACATGC 131 CYA_BA_1003_1025_R CCACTTTTAATAAGGTTTGTAGC 76834 INFB_EC_1365_1393_F TGCTCGTGGTGCACAAGTAACGGATA 524INFB_EC_1439_1467_R TGCTGCTTTCGCATGGTTAATTGCTTCA 1248 TTA A 35LEF_BA_1033_1052_F TCAAGAAGAAAAAGAGC 254 LEG_BA_1119_1135_RGAATATCAATTTGTAGC 803 36 LEF_BA_1036_1066_F CAAGAAGAAAAAGAGCTTCTAAAAAG44 LEF_BA_1119_1149_R AGATAAAGAATCACGAATATCAATTTGT 745 AATAC AGC 37LEF_BA_756_781_F AGCTTTTGCATATTATATCGAGCCAC 26 LEF_BA_843_872_RTCTTCCAAGGATAGATI2TATTTCTTGTT 1135 CG 38 LEF_BA_758_778_FCTTTTGCATATTATATCGAGC 90 LEF_BA_843_865_R AGGATAGATTTATTTCTTGTTCG 748 39LEF_BA_795_813_F TTTACAGCTTTATGCACCG 700 LEF_BA_883_900_RTCTTGACAGCATCCGTTG 1140 40 LEG_BA_883_899_F CAACGGATGCTGGCAAG 43LEF_BA_939_958_R CAGATAAAGAATCGCTCCAG 762 41 PAG_BA_122_142_FCAGAATCAAGTTCCCAGGGG 49 PAG_BA_190_209_R CCTGTAGTAGAAGAGGTAAC 781 42PAG_BA_123_145_F AGAATCAAGTTCCCAGGGGTTAC 22 PAG_BA_187_210 RCCCTGTAGTAGAAGAGGTAACCAC 774 43 PAG_BA_269_287_F AATCTGCTATTTGGTCAGG 11PAG_BA_326_344_R TGATTATCAGCGGAAGTAG 1186 44 PAG_BA_655_675_FGAAGGATATACGGTTGATGTC 93 PAG_BA_755_772_R CCGTGCTCCATTTTTCAG 778 45PAG_BA_753_772_F TCCTGAAAAATGGAGCACGG 341 PAG_BA_849_868_RTCGGATAAGCTGCCACAAGG 1089 46 PAG_BA_763_761_F TGGAGCACGGCTTCTGATC 552PAG_BA_849_868_R TCGGATAAGCTGCCACAAGG 1089 47 RPOC_EC_1018_1045_FCAAAACTTATTAGGTAAGCGTGTTGA 39 RPOC_EC_1095_1124_RTCAAGCGCCATTTCTTTTGGTAAACCAC 959 CT AT 48 RPOC_EC_1018_1045_2_FCAAAACTTATTAGGTAAGCGTGTTGA 39 PROC_EC_1095_1124_2_RTCAAGCGCCATCTCTTTCGGTAATCCAC 958 CT AT 49 RPOC_EC_114_140_FTAAGAAGCCGGAAACCATCAACTACC 158 RFOC_EC_213_232_R GGCGCTTGTACTTACCGCAC831 G 50 RFOC_EC_2178_2196_F TGATTCTGGTGCCCGTGGT 478 RFOC_EC_2225_2246_RTTGGCCATCAGGCCACGCATAC 1414 51 RFOC_EC_2178_2196_2_F TGATTCCGGTGCCCGTGGT477 RFOC_EC_2225_2246_2_R TTGGCCATCAGACCACGCATAC 1413 52RFOC_EC_2218_2241_F CTGGCAGGTATGCGTGGTCTGATG 81 RFOC_EC_2313_2337_RCGCACCGTGGGTTGAGATGAAGTAC 790 53 RFOC_EC_2218_2241_2_FCTTGCTGGTATGCGTGGTCTGATG 86 EFOC_EC_2313_2337_2_RCGCACCATGCGTAGAGATGAAGTAC 789 54 EPOC_EC_808_833_FCGTCGGGTGATTAACCGTAACAACCG 75 RPOC_EC_865_889_RGTTTTTCGTTGCGTACGATGATGTC 847 55 RFOC_EC_808_833_2_FCGTCGTGTAATTAACCGTAACAACCG 76 RFOC_EC_865_891_RACGTTTTTCGTTTTGAACGATAATGCT 741 56 RFOC_EC_993_1019_FCAAAGGTAAGCAAGGTCGTTTCCGTC 41 RFOC_EC_1036_1059_RCGAACGGCCTGAGTAGTCAACACG 785 A 57 RPOC_EC_993_1019_2_FCAAAGGTAAGCAAGGACGTTTCCGTC 40 RPOC_EC_1036_1059_2_RCGAACGGCCAGAGTAGTCAACACG 784 A 58 SSPE_BA_115_137_FCAAGCAAACGCACAATCAGAAGC 45 SSPE_BA_197_222_R TGCACGTCTGTTTCAGTTGCAAATTC1201 59 TUFB_EC_239_259_F TAGACTGCCCAGGACACGCTG 204 TUFB_EC_283_303_RGCCGTCCATCTGAGCAGCACC 815 60 TUFB_EC_239_259_2_F TTGACTGCCCAGGTCACGCTG678 TUFB_EC_283_303_2_R GCCGTCCATTTGAGCAGCACC 816 61 TUFB_EC_976_1000_FAACTACCGTCCCCAGTTCTACTTCC 4 TUFB_EC_1045_1068_R GTTGTCCCCAGGCATAACCATTTC845 62 TUFB_EC_976_1000_2_F AACTACCGTCCTCAGTTCTACTTCC 5TUFB_EC_1045_1068_2_R GTTGTCACCAGCCATTACCATTTC 844 63 TUFB_EC_985_1012_FCCACAGTTCTACTTCCGTACTACTGA 56 TUFB_EC_1033_1062_RTCCAGGCATTACCATTTCTACTCCTTCT 1006 CG CG 66 RPLB_EC_650_679_FGACCTACAGTAAGAGGTTCTGTAATG 98 RPLB_EC_739_762_R TCCAACTGCTCCTTTACCCCATGG999 AACC 67 RPLB_EC_688_710_F CATCCACACGGTGGTGGTGAAGG 54RPLB_EC_736_757_R GTGCTGGTTTACCCCATGGAGT 842 68 RPOC_EC_1036_1060_FCGTGTTGACTATTCGGGGCGTTCAG 78 RPOC_EC_1097_1126_RATTCAACAGCCATTTCTTTTGGTAAACC 754 AC 69 RFOB_EC_3762_3790_FTCAACAACCTCTTCGAGGTAAAGCTC 248 RPOB_EC_3836_3865_RTTTCTTGAAGAGTATGAGCTGCTCCGTA 1435 AGT AG 70 RPLB_EC_688_710_FCATCCACACGGTGGTGGTGAAGG 54 RPLB_EC_743_771_RTGTTTTGTATCCAAGTCCTGGTTTACCC 1356 C 71 VALS_EC_1105_1124_FCGTGGCGGCGTGGTTATCGA 77 VALS_EC_1195_1218_R CGGTACGAACTGGATGTCGCCGTT 79572 RFOB_EC_1845_1866_F TATCGCTCAGGCGAACTCCAAC 233 RPOB_EC_1909_1929_RGCTGGATTCGCCTTTGCTACG 825 73 RPLB_EC_669_698_FTGTAATGAACCCTAATGACCATCCAC 623 RPLB_EC_735_761_RCCAAGTGCTGGTTTACCCCATGGAGTA 767 ACGG 74 RPLB_EC_671_700_FTAATGAACCCTAATGACCATCCACAC 169 RPLB_EC_737_762_RTCCAAGTGCTGGTTTACCCCATGGAG 1000 GGTG 75 SP101_SPET11_1_29_FAACCTTAATTGGAAAGAAACCCAAGA 2 SP101_SPET11_92_116_RCCTACCCAACGTTCACCAAGGGCAG 779 AGT 76 SP101_SPET11_118_14GCTGGTGAAAATAACCCAGATGTCGT 115 SP101_SPET11_213_238_RTGTGGCCGATTTCACCACCTGCTCCT 1340 7_F CCTC 77 SP101_SPET11_216_24AGCAGGTGGTGAAATCGGCCACATGA 24 SP101_SPET11_308_333_RTGCCACTTTGACAACTCCTGTTGCTG 1209 3_F TT 78 SP101_SPET11_226_29CTTGTACTTGTGGCTCACACGGCTGT 89 SP101_SPET11_355_380_RGCTGCTTTGATGGCTGAATCCCCTTC 824 5_F TTGG 79 SP101_SPET11_322_34GTCAAAGTGGCACGTTTACTGGC 132 SP101_SPET11_423_441_R ATCCCCTGCTTCTGCTGCC753 4_F 80 SP101_SPET11_358_38 GGGGATTCAGCCATCAAAGCAGCTAT 126SP101_SPET11_448_473_R CCAACCTTTTCCACAACAGAATCAGC 766 7_F 81SP101_SPET11_600_62 CCTTACTTCGAACTATGAATCTTTTG 62 SP101_SPET11_686_714_RCCCATTTTTTCACGCATGCTGAAAATAT 772 9_F GAAG C 82 SP101_SPET11_658_68GGGGATTGATATCACCGATAAGAAGA 127 SP101_SPET11_756_784_RGATTGGCGATAAAGTGATATTTTCTAAA 813 4_F A A 83 SP101_SPET11_776_80TCGCCAATCAAAACTAAGGGAATGGC 364 SP101_SPET11_871_896_RGCCCACCAGAAAGACTAGCAGGATAA 814 1_F 84 SP101_SPET11_893_92_GGGCAACAGCAGCGGATTGCGATTGC 123 SP101_SPET11_988_1012_RCATGACAGCCAAGACCTCACCCACC 763 1_F GCG 85 SP1010_SPET11_1154_1CAATACCGCAACAGCGGTGGCTTGGG 47 SP101_SPET11_1251_1277_RGACCCCAACCTGGCCTTTTGTCGTTGA 804 179_F 86 SP101_SPET11_1314_1CGCAAAAAAATCCAGCTATTAGC 68 SP101_SPET11_1403_1431_RAAACTATTTTTTTAGCTATACTCGAACA 711 336_F C 87 SP101_SPET11_1408_1CGAGTATAGCTAAAAAAATAGTTTAT 67 SP101SPET11_486_1515_RGGATAATTGGTCGTAACAGGGATAGTG 828 437_F GACA AG 88 SP101_SPET11_1688_1CCTATATTAATCGTTTACAGAAACTG 60 SP101_SPET11_1783_1808_RATATGATTATCATTGAACTGCGGCCG 752 716_F GCT 89 SP101_SPET11_1711_1CTGGCTAAAACTTTGGCAACGGT 82 SP101_SPET11_1808_1835_RGCGTGACGACCTTCTTGAATTGTAATCA 821 733_F 90 SP101_SPET11_1807_1ATGATTACAATTCAAGAAGGTCGTCA 33 SP101_SPET11_1901_1927_RTTGGACCTGTAATCAGCTGAATACTGG 1412 835_F CGC 91 SP101_SPET11_1967_1TAACGGTTATCATGGCCCAGATGGG 155 SP101_SPET11_2062_2083_RATTGCCCAGAAATCAAATCATC 755 991_F 92 SP101_SPET11_2260_2CAGAGACCGTTTTATCCTATCAGC 50 SP101_SPET11_2375_2397_RTCTGGGTGACCTGGTGTTTTAGA 1131 283_F 93 SP101_SPET11_2375_2TCTAAAACACCAGGTCACCCAGAAG 390 SP101_SPET11_2470_2497_RAGCTGCTAGATGAGCTTCTGCCATGGCC 747 399_F 94 SP101_SPET11_2468_2ATGGCCATGGCAGAAGCTCA 35 SP101_SPET11_2543_2570_RCCATAAGGTCACCGTCACCATTCAAAGC 770 487_F 95 SP101_SPET11_2961_2ACCATGACACAAGGCATTTTGACA 15 SP101_SPET11_3023_3045_RGGAATTTACCAGCGATAGACACC 827 984_F 96 SP101_SPET11_3075_3GATGACTTTTTAGCTAATGGTCAGGC 108 SP101_SPET11_3168_3196_RAATCGACGACCATCTTGGAAAGATTTCT 715 103_F AGC C 97 SP101_SPET11_3386_3AGCGTAAAGGTGAACCTT 25 SP101_SPET11_3480_3506_RCCAGCAGTTACTGTCCCCTCATCTTTG 769 403_F 98 SP101_SPET11_3511_3GCTTCAGGAATCAATGATGGAGCAG 116 SP101_SPET11_3605_3629_RGGGTCTACACCTGCACTTGCATAAC 832 535_F 111 RPOB_EC_3775_3803_FCTTGGAGGTAAGTCTCATTTTGGTGG 87 RPOB_EC_3829_3858_RCGTATAAGCTGCACCATAAGCTTGTAAT 797 GCA GC 112 VALS_EC_1833_1850_FCGACGCGCTGCGCTTCAC 65 VALS_EC_1920_1943_R GCGTTCCACAGCTTGTTGCAGAAG 822113 RPOB_SC_1336_1353_F GACCACCTCGGCAACCGT 97 RPOB_EC_1438_1455_RTTCGCTCTCGGCCTGGCC 1386 114 TUFB_EC_225_251_F GCACTATGCACACGTAGATTGTCCTG111 TUFB_EC_284_309_R TATAGCACCATCCATCTGAGCGGCAC 930 G 115DNAK_EC_428_449_F CGGCGTACTTCAACGACAGCCA 72 DNAK_EC_503_522_RCGCGGTCGGCTCCTTGATGA 792 116 VALS_EC_1920_1943_FCTTCTGCAACAAGCTGTGGAACGC 85 VALS_EC_1948_1970_R TCGCAGTTCATCACCACGAAGCG1075 117 TUFB_EC_757_774_F AAGACGACCTGCACGGGC 6 TUFB_EC_849_867_RGCGCTCCACCTCTTCACGC 819 118 23S_EC_2646_2667_F CTGTTCTTAGTACGAGAGGACC 8423S_EC_2745_2765_R TTCCTGCTTACATCCTTTCAC 1389 119 16S_EC_969_985_1P_FACGCGAAGAACCTTACpC 19 16S_EC_1061_1078_2P_R ACGACACGAGCpTpGACGAC 733 12016S_EC_972_985_2P_F CGAAGAACpCpTTACC 63 16S_SC_1064_1075_2P_RACACGAGCpTpGAC 727 121 16S_EC_972_985_F CGAAGAACCTTACC 6316S_EC_1064_1075_R ACACGAGCTGAC 727 122 TRNA_ILE- CCTGATAAGGGTGAGGTCG 6123S_EC_40_59_R ACGTCCTTCATCGCCTCTGA 740 RRNG_EC_32_50.2_F 12323S_EC_−7_15_F GTTGTGAGGTTAAGCGACTAAG 140 23S_EC_430_450_RCTATCGGTCAGTCAGGAGTAT 799 124 23S_EC_−7_15_F GTTGTGAGGTTAAGCGACTAAG 14123S_EC_891_910_R TTGCATCGGGTTGGTAAGTC 1403 125 23S_EC_430_450_FATACTCCTGACTGACCGATAG 30 23S_EC_1424_1442_R AACATAGCCTTCTCCGTCC 712 12623S_EC_891_910_F GACTTACCAACCCGATGCAA 100 23S_EC_1908_1931_RTACCTTAGGACCGTTATAGTTACG 893 127 23S_EC_1424_1442_F GGACGGAGAAGGCTATGTT117 23S_EC_2475_2494_R CCAAACACCGCCGTCGATAT 765 128 23S_EC_1908_1931_FCGTAACTATAACGGTCCTAAGGTA 73 23S_EC_2833_2852_R GCTTACACACCCGGCCTATC 826129 23S_EC_2475_2494_F ATATCGACGGCGGTGTTTGG 31 TRNA_ASP-GCGTGACAGGCAGGTATTC 820 RRNh_EC_23_41.2_R 131 16S_EC_−60_−39_FAGTCTCAAGAGTGAACACGTAA 28 16S_EC_508_525_R GCTGCTGGCACGGAGTTA 823 13216S_EC_326_345_F GACACGGTCCAGACTCCTAC 95 16S_EC_1041_1058_RCCATGCAGCACCTGTCTC 771 133 16S_EC_705_724_F GATCTGGAGGAATACCGGTG 10716S_EC_1493_1512_R ACGGTTACCTTGTTACGACT 739 134 16S_EC_1268_1287_FGAGAGCAAGCGGACCTCATA 101 TRNA_ALA- CCTCCTGCGTGCAAAGC 780RENH_EC_30_46.2_R 135 16S_EC_969_985_F ACGCGAAGAACCTTACC 1916S_EC_1061_1078.2_R ACAACACGAGCTGACGAC 719 137 16S_EC_969_985_FACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_I14_R ACAACACGAGCTGICGAC 721 13816S_EC_969_985_F ACGCOAAGAACCTTACC 19 16S_EC_1061_1078.2_I12_RACAACACGAGCTGACGAC 718 139 16S_EC_969_985_F ACGCGAACAACCTTACC 1916S_EC_1061_1078.2_I11_R ACAACACGAGITGACGAC 722 140 16S_EC_969_985_FACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_I16_R ACAACACGAGCTGACIAC 720 14116S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_2I_RACAACACGAICTIACGAC 723 142 16S_EC_969_985_F ACGCGAAGAACCTTACC 1916S_EC_1061_1078.2_3I_R ACAACACIAICTIACGAC 724 143 16S_EC_969_985_FACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_4I_R ACAACACIAICTIACIAC 725 14723S_EC_2652_2669_F CTAGTACGACAGGACCGG 79 23S_EC_2741_2760_RACTTAGATGCTTTCAGCGGT 743 158 16S_EC_683_700_F GTGTAGCGGTGAAATGCG 13716S_EC_880_894_R CGTACTCCCCAGGCG 796 159 16S_EC_1100_1116_FCAACGACCGCAACCCTT 42 16S_EC_1174_1188_R TCCCCACCTTCCTCC 1019 21SSSPE_BA_121_137_F AACGCACAATCAGAAGC 3 SSPE_BA_197_216_RTCTGTTTCASTTGCAAATTC 1132 220 GROL_EC_941_959_F TGGAAGATCTGGGTCAGGC 544GROL_EC_1039_1060_R CAATCTGCTGACGGATCTGAGC 759 221 INFB_EC_1103_1124_FGTCGTGAAAACGAGCTGGAAGA 133 INFB_EC_1174_1191_R CATGATGGTCACAACCGG 764222 HFLB_EC_1082_1102_F TGGCGAACCTGGTGAACGAAGC 569 HFLB_EC_1144_1168_RCTTTCGCTTTCTCGAACTCAACCAT 802 223 INFB_EC_1969_1994_FCGTCAGGGTAAATTCCGTGAAGTTAA 74 INFB_EC_2038_2058_R AACTTCGCCTTCGGTCATGTT713 224 GROL_EC_219_242_F GGTGAAAGAAGTTGCCTCTAAAGC 128 GROL_EC_328_350_RTTCAGGTCCATCGGGTTCATGCC 1377 225 VALS_EC_1105_1124_FCGTGGCGGCGTGGTTATCGA 77 VALS_EC_1195_1214_R ACGAACTGGATGTCGCCGTT 732 22616S_EC_556_575_F CGGAATTACTGGGCGTAAAG 70 16S_EC_683_700_RCGCATTTCACCGCTACAC 791 227 RPOC_EC_1256_1277_F ACCCAGTGCTGCTGAACCGTGC 16RPOC_EC_1295_1315_R GTTCAAATGCCTGGATACCCA 843 228 16S_EC_774_795_FGGGAGCAAACAGGATTAGATAC 122 16S_EC_880_894_R CGTACTCCCCAGGCG 796 229RPOC_EC_1584_1604_F TGGCCCGAAAGAAGCTGAGCG 567 RPOC_EC_1623_1643_RACGCGGGCATGCAGAGATGCC 737 230 16S_EC_1082_1100_F ATGTTGGGTTAAGTCCCGC 3716S_EC_1177_1196_R TGACGTCATCCCCACCTTCC 1158 231 16S_EC_1389_1407_FCTTGTACACACCGCCCGTC 88 16S_EC_1525_1541_R AAGGAGGTGATCCAGCC 714 23216S_EC_1303_1323_F CGGATTGGAGTCTGCAACTCG 71 16S_EC_1389_1407_RGACGGGCGGTGTGTACAAG 808 233 23S_EC_23_37_F GGTGGATGCCTTGGC 12923S_EC_115_130_R GGGTTTCCCCATTCGG 833 234 23S_EC_187_207_FGGGAACTGAAACATCTAAGTA 121 23S_EC_242_256_R TTCGCTCGCCGCTAC 1385 23S23S_EC_1602_1620_F TACCCCAAACCGACACAGG 184 23S_EC_1686_1703_RCCTTCTCCCGAAGTTACG 782 236 23S_EC_1685_1703_F CCGTAACTTCGGGAGAAGG 5823S_EC_1828_1842_R CACCGGGCAGGCGTC 760 237 23S_EC_1827_1843_FGACGCCTGCCCCGTGC 99 23S_EC_1929_1949_R CCGACAAGGAATTTCGCTACC 775 23823S_EC_2434_2456_F AAGGTACTCCGGGGATAACAGGC 9 23S_EC_2490_2511_RAGCCGACATCGAGGTGCCAAAC 746 239 23S_EC_2599_2616_F GACAGTTCCGTCCCTATC 9623S_EC_2653_2669_R CCGGTCCTCTCGTACTA 777 240 23S_EC_2653_2669_FTAGTACGAGAGGACCGG 227 23S_EC_2737_2758_R TTAGATGCTTTCACCACTTATC 1369 24123S_BS_−68_−44_F AAACTAGATAACAGTAGACATCAC 1 23S_EC_5_21_RGTGCGCCCTTTCTAACTT 841 242 16S_EC_8_27_F ACAGTTTGATCATGGCTCAG 2316S_EC_342_358_R ACTGCTGCCTCCCGTAG 742 243 16S_EC_314_332_FCACTGGAACTGAGACACGG 48 16S_EC_556_575_R CTTTACGCCCAGTAATTCCG 801 24416S_EC_518_536_F CCAGCAGCCCCGGTAATAC 57 16S_EC_774_795_RGTATCTAATCCTGTTTGCTCCC 839 245 16S_EC_683_700_F GTGTAGCGGTGAAATGCG 13716S_EC_967_985_R CGTAAGGTTCTTCGCGTTG 835 246 16S_EC_937_954_FAAGCGGTGGAGCACGTGG 7 16S_EC_1220_1240_R ATTGTAGCACGTGTGTAGCCC 757 24716S_EC_1195_1213_F CAAGTCATCATGGCCCTTA 46 16S_EC_1525_1541_5AAGGAGGTGATCCAGCC 714 248 16S_EC_8_27_F AGAGTTTGATCATGGCTCAG 2316S_EC_1525_1541_R AAGGAGGTGATCCAGCC 714 249 23S_EC_1831_1849_FACCTGCCCAGTGCTGGAAG 18 23S_EC_1919_1936_R TCGCTACCTTAGGACCGT 1080 25016S_EC_1387_1407_F GCCTTGTACACACCTCCCGTC 112 16S_EC_1494_1513_RCACGGCTACCTTGTTACGAC 761 251 16S_EC_1390_1411_F TTGTACACACCGCCCGTCATAC693 A6S_EC_1486_1505_R CCTTGTTACGACTTCACCCC 783 252 16S_EC_1367_1387_FTACGGTGAATACGTTCCCGGG 191 16S_EC_1485_1506_R ACCTTGTTACGACTTCACCCCA 731253 16S_EC_804_822_F ACCACGCCGTAAACGATGA 14 16S_EC_909_929_RCCCCCGTCAATTCCTTTGAGT 773 254 16S_EC_791_812_F GATACCCTGGTAGTCCACACCG106 16S_EC_886_904_R GCCTTGCGACCGTACTCCC 817 255 16S_EC_789_810_FTAGATACCCTGCTAGTCCACGC 206 16S_EC_882_899_R GCGACCGTACTCCCCAGG 818 25616S_EC_1092_1109_F TAGTCCCGCAACGAGCGC 228 16S_EC_1174_1198_RCACGTCATCCCCACCTTCCTCC 810 257 23S_EC_2586_2607_F TAGAACGTCGCCAGACAGTTCG203 23S_EC_2658_2677_R AGTCCATCCCGGTCCTCTCG 749 258 RNASEP_SA_31_49_FGAGGAAACTCCATGCTCAC 103 RNASEP_SA_358_379_R ATAAGCCATGTTCTGTTCCATC 750258 RNASEP_SA_31_49_F GAGGAAAGTCCATGCTCAC 103 RNASEP_EC_345_362_RATAAGCCGGGTTCTGTCG 751 258 RNASEP_SA_31_49_F GAGGAAAGTCCATGCTCAC 103RNASEP_ES_363_384_R GTAAOCCATGTTTTGTTCCATC 838 258 RNASEP_ES_43_61_FGACGAAAGTCCATGCTCCC 104 RNASEP_SA_358_379_R ATAAGCCATGTTCTGTTCCATC 750258 RNASEP_ES_43_61_F CAGGAAAGTCCATGCTCGC 104 RNASEP_EC_345_362_RATAAGCCGGGTTCTGTCG 751 258 RNASEP_ES_43_61_F GAGGAAAGTCCATGCTCGC 104RNASEP_ES_363_384_R GTAAGCCATGTTTTGTTCCATC 838 258 RNASEP_EC_61_277_FGAGGAAAGTCCGGGCTC 105 RNASEP_SA_358_379_R ATAAGCCATGTTCTCTTCCATC 750 258RNASEP_EC_61_77_F GAGGAAAGTCCGGCCTC 105 RNASEP_EC_345_362_RATAAGCCGGGTTCTGTCG 751 258 RNASEP_EC_61_277_F GAGGAAAGTCCGGGCTC 105RNASEP_ES_363_384_R GTAAGCCATGTTTTGTTCCATC 838 259 RNASEP_ES_43_61_FGAGCAAAGTCCATGCTCGC 104 RNASEP_ES_363_384_R GTAAGCCATGTTTTGTTCCATC 838260 RNASEP_EC_61_77_F GAGGAAAGTCCGGGCTC 105 RNASEP_EC_345_362_RATAAGCCGGGTTCTGTCG 751 262 RNASEP_SA_31_49_F GAGGAAACTCCATGCTCAC 103RNASEP_SA_358_379_R ATAAGCCATGTTCTGTTCCATC 750 263 16S_EC_1082_1100_FATGTTGGGTTAAGTCCCGC 37 16S_EC_1525_1541_R AAGGAGGTGATCCAGCC 714 26416S_EC_556_575_F CGGAATTACTGCGCGTAAAG 70 16S_EC_774_795_RGTATCTAATCCTGTTTGCTCCC 839 265 16S_EC_1082_1100_F ATGTTGGGTTAAGTCCCGC 3716S_EC_1177_1196_10G_R TGACGTCATGCCCACCTTCC 1160 266 16S_EC_1082_1100_FATGTTGCCTTAACTCCCGC 37 16S_EC_1177_1196_10G_115_R TGACGTCATGGCCACCTTCC1161 268 YAED_EC_513_532_F_M GGTGTTAAATAGCCTGGCAG 130 TRNA_ALA-AGACCTCCTGCGTGCAAAGC 744 OD RRNH_EC_30_49_F_MOD 269 16S_EC_1082_1100_F_ATGTTCGGTTAAGTCCCGC 37 16S_EC_1177_1196_R_MOD TGACGTCATCCCCACCTTCC 1158MOD 270 23S_EC_2586_2607_F TAGAACGTCGCGAGACAGTTCG 20323S_EC_2658_2677_R_MOD AGTCCATCCCGGTCCTCTCG 749 MOD 272 16S_EC_969_985_FACCCGAAGAACCTTACC 19 16S_EC_1389_1407_R GACGGGCCGTGTGTACAAG 807 27316S_EC_683_700_F GTGTAGCGGTGAAATGCG 137 16S_EC_1303_1323_RCGAGTTGCAGACTGCGATCCG 788 274 16S_EC_49_68_F TAACACATGCAAGTCGAACG 15216S_EC_880_894_R CGTACTCCCCAGGCG 796 275 16S_EC_49_68_FTAACACATGCAAGTCGAACG 152 16S_EC_1061_1078_R ACGACACGAGCTGACGAC 734 277CYA_BA_1349_1370_F ACAACGAAGTACAATACAAGAC 12 CYA_BA_1426_1447_RCTTCTACATTTTAGCCATCAC 800 278 16S_EC_1090_1111_2_FTTAAGTCCCGCAACGAGCGCAA 650 16S_EC_1175_1196_R TGACGTCATCCCCACCTTCCTC1159 279 16S_EC_405_432_F TGAGTGATGAAGGCCTTAGGGTTGTA 46416S_EC_507_527_R CGGCTGCTGGCACGAAGTTAG 793 AA 280 GROL_EC_496_518_FATGGACAAGGTTGGCAAGGAAGG 34 GROL_EC_577_596_R TAGCCGCGGTCGAATTGCAT 914281 GROL_EC_511_536_F AAGGAAGGCGTGATCACCGTTGAAGA 8 GROL_EC_571_593_RCCGCGGTCGAATTGCATGCCTTC 776 288 RPOB_EC_3802_3821_F CAGCGTTTCGGCGAAATGGA51 RPOB_EC_3862_3885_R CGACTTGACGGTTAACATTTCCTG 786 289RPOB_EC_3799_3821_F GGGCAGCGTTTCGGCGAAATGGA 124 RPOB_EC_3862_3888_RGTCCGACTTGACGGTCAACATTTCCTG 840 290 RPOC_EC_2146_2174_FCAGGAGTCGTTCAACTCGATCTACAT 52 RPOC_EC_2227_2245_R ACGCCATCAGGCCACGCAT736 GAT 291 ASPS_EC_405_422_F GCACAACCTGCGGCTGCG 110 ASPS_EC_521_538_RACGGCACGAGGTAGTCGC 738 292 RPOC_EC_1374_1393_F CGCCGACTTCGACGGTGACC 69RPOC_EC_1437_1455_R GAGCATCAGCGTGCGTGCT 811 293 TUFB_EC_957_979_FCCACACGCCGTTCTTCAACAACT 55 TUFB_EC_1034_1058_R GGCATCACCATTTCCTTGTCCTTCG829 294 16S_EC_733_F GAGAGTTTGATCCTGGCTCAGAACGA 102 16S_EC_101_122_RTGTTACTCACCCGTCTGCCACT 1345 A 295 VALS_EC_610_649_F ACCGAGCAAGGAGACCAGC17 VALS_EC_705_727_R TATAACGCACATCGTCAGGGTGA 929 344 16S_EC_971_990_FGCGAAGAACCTTACCAGGTC 113 16S_EC_1043_1062_R ACAACCATGCACCACCTGTC 726 34616S_EC_713_732_TMOD_F TAGAACACCGATGGCGAAGGC 202 16S_EC_789_809_TMOD_RTCGTGGACTACCAGGGTATCTA 1110 347 16S_EC_785_806_TMOD_FTGGATTAGAGACCCTGGTAGTCC 560 16S_EC_880_897_TMOD_R TGGCCGTACTCCCCAGGCG1278 348 16S_EC_960_981_TMOD_F TTTCGATGCAACGCGAAGAACCT 70616S_EC_1054_1073_TMOD_R TACGAGCTGACGACAGCCATG 895 34923S_BC_1826_1843_TM TCTGACACCTGCCCGGTGC 401 23S_EC_1906_1924_TMOD_RTGACCGTTATAGTTACGGCC 1156 OD_F 350 CAPC_BA_274_303_TMOTGATTATTGTTATCCTGTTATGCCAT 476 CAPC_BA_349_376_TMOD_RTGTAACCCTTGTCTTTGAATTGTATTTG 1314 D_F TTGAG C 351 CYA_BA_1353_1379_TMTCGAAGTACAATACAAGACAAAAGAA 355 CYA_BA_1448_1467_TMOD_RTTGTTAACGGCTTCAAGACCC 1423 OD_F GG 352 INFB_EC_1365_1393_TTTGCTCGTGGTGCACAAGTAACGGAT 687 INFB_EC_1439_1467_TMOD_RTTGCTGCTTTCGCATGGTTAATTGCTTC 1411 MOD_F AA 353 LEF_BA_756_781_TMOD_FTAGCTTTTGCATATTATATCGAGCCA 220 LEF_BA_843_872_TMOD_RTTCTTCCAAGGATAGATTTATTTCTTGT 1394 C TCG 354 RPOC_EC_2218_2241_TTCTGGCAGGTATGCGTGGTCTGATG 405 RPOC_EC_2313_2337_TMOD_RTCOCACCGTGGGTTGAGATGAAGTAC 1072 MOD_F 355 SSPE_BA_115_137_TMOTCAAGCAAACGCACAATCAGAAGC 255 SSPE_BA_197_222_TMOD_RTTGCACGTCTGTTTCAGTTGCAAATTC 1402 D_F 356 RPLB_EC_650_679_TMOTGACCTACAGTAAGAGGTTCTGTAAT 449 RFLB_EC_739_762_TMOD_RTTCCAAGTGCTGGTTTACCCCATGG 1380 D_F GAACC 357 RPLB_EC_688_710_TMOTCATCCACACGGTGGTGGTGAAGG 296 RFLB_EC_736_757_TMOD_RTGTGCTGGTTTACCCCATGGAGT 1337 D_F 358 VALS_EC_1105_1124_TTCGTGGCGGCGTGGTTATCGA 385 VALS_EC_1195_1218_TMOD_RTCGGTACGAACTGGATGTCGCCGTT 1093 MOD_F 359 RPOB_EC_1845_1866_TTTATCGCTCAGGCGAACTCCAAC 659 RPOB_EC_1909_1929_TMOD_RTGCTGGATTCGCCTTTGCTACG 1250 MOD_F 360 23S_EC_2646_2667_TMTCTGTTCTTAGTACGACAGGACC 409 23S_EC_2745_2765_TMOD_RTTTCGTGCTTAGATCCTTTCAG 1434 OD_F 361 16S_EC_1090_1111_2_TTTAAGTCCCGCAACGAGCGCAA 697 16S_EC_1175_1196_TMOD_RTTGACCTCATCCCCACCTTCCTC 1398 TMOD_F 362 RFOB_EC_3799_3821_TTGGCCAGCGTTTCGGCGAAATGGA 581 RPOB_EC_3862_3888_TMOD_RTGTCCGACTTGACGGTCAACATTTCCTG 1325 MOD_F 363 RPOC_EC_2146_2174_TTCAGGAGTCGTTCAACTCGATCTACA 284 EFOC_EC_2227_2245_TMOD_RTACCCCATCAGGCCACCCAT 898 MOD_F 364 RPOC_EC_1374_1393_TTCGCCGACTTCGACGGTGACC 367 RPOC_EC_1437_1455_TMOD_R TGAGCATCAGCGTGCGTGCT1166 MOD_F 367 TUFB_EC_957_979_TMO TCCACACGCCGTTCTTCAACAACT 308TUFB_EC_1034_1058_TMOD_R TGGCATCACCATTTCCTTGTCCTTCG 1276 D_F 423SP101_SPET11_893_92 TGGGCAACAGCAGCGGATTGCGATTG 580SP101_SPET11_988_1012_TMO TCATGACAGCCAAGACCTCACCCACC 990 1_TMOD_F CGCGD_R 424 SP101_SPET11_1154_1 TCAATACCGCAACAGCGGTGGCTTGG 258SP101_SPET11_1251_1277_TM TGACCCCAACCTGGCCTTTTGTCGTTCA 1155 179_TMOD_F GOD_R 425 SP101_SPET11_118_14 TGCTGGTGAAAATAACCCAGATGTCG 528SP101_SPET11_213_238_TMOD_ TTGTGGCCGATTTCACCACCTGCTCCT 1422 7_TMOD_FTCTTC R 426 SP101_SPET11_1314_1 TCGCAAAAAAATCCAGCTATTAGC 363SP101_SPET11_1403_1431_TM TAAACTATTTTTTTAGCTATACTCGAAC 849 336_TMOD_FOD_R AC 427 SP101_SPET11_1408_1 TCGAGTATAGCTAAAAAAATAGTTTA 359SP101_SPET11_1486_1515_TM TGGATAATTGGTCGTAACAAGGGATAGT 1268 437_TMOD_FTGACA OD_R GAG 428 SP101_SPET11_1688_1 TCCTATATTAATCGTTTACAGAAACT 334SP101_SPET11_1783_1808_TM TATATGATTATCATTGAACTGCGGCCG 932 716_TMOD_FGGCT OD_R 429 SP101_SPET11_1711_1 TCTGGCTAAAACTTTGGCAACGGT 406SP101_SPET11_1808_1835_TM TGCGTGACGACCTTCTTGAATTGTAATC 1239 733_TMOD_FOD_R A 430 SP101_SPET11_1807_1 TATGATTACAATTCAAGAAGGTCGTC 235SP101_SPET11_1901_1927_TM TTTGGACCTGTAATCAGCTGAATACTGG 1439 835_TMOD_FACGC OD_R 431 SP101_SPET11_1967_1 TTAACGGTTATCATGGCCCAGATGGG 649SP101_SPET11_2062_2083_TM TATTGCCCAGAAATCAAATCATC 940 991_TMOD_F OD_R432 SP101_SPET11_216_24 TAGCAGGTGGTGAAATCGGCCACATG 210SP101_SPET11_308_333_TMOD_ TTGCCACTTTGACAACTCCTGTTGCTG 1404 3_TMOD_F ATTR 433 SP101_SPET11_2260_2 TCAGAGACCGTTTTATCCTATCAGC 272SP101_SPET11_2375_2397_TM TTCTGGGTGACCTGGTGTTTTAGA 1393 283_TMOD_F OD_R434 SP101_SPET11_2375_2 TTCTAAAACACCAGGTCACCCAGAAG 675SP101_SPET11_2470_2497_TM TAGCTGCTAGATGAGCTTCTGCCATGGC 918 399_TMOD_FOD_R C 435 SP101_SPET11_2468_2 TATGGCCATGGCAGAAGCTCA 238SP101_SPET11_2543_2570_TM TCCATAAGGTCACCGTCACCATTCAAAG 1007 487_TMOD_FOD_R C 436 SP101_SPET11_266_29 TCTTGTACTTGTGGCTCACACGGCTG 417SP101_SPET11_355_380_TMOD_ TGCTGCTTTGATGGCTGAATCCCCTTC 1249 5_TMOD_FTTTGG R 437 SP101_SPET11_2961_2 TACCATGACAGAAGGCATTTTGACA 183SP101_SPET11_3023_3045_TM TGGAATTTACCAGCGATAGACACC 1264 984_TMOD_F OD_R438 SP101_SPET11_3075_3 TGATGACTTTTTAGCTAATGGTCAGG 473SP101_SPET11_3168_3196_TM TAATCGACGACCATCTTGGAAAGATTTC 875 103_TMOD_FCAGC OD_R TC 439 SP101_SPET11_322_34 TGTCAAAGTGGCACCTTTACTGGC 631SP101_SPET11_423_441_TMOD_ TATCCCCTGCTTCTGCTGCC 934 4_TMOD_F R 440SP101_SPET11_3368_3 TAGCGTAAAGGTGAACCTT 215 SP101_SPET11_3480_3506_TMTCCACCAGTTACTCTCCCCTCATCTTTC 1005 403_TMOD_F OD_R 441SP101_SPET11_3511_3 TGCTTCAGCAATCAATGATGGAGCAG 531SP101_SPET11_3605_3629_TM TGGGTCTACACCTGCACTTCCATAAC 1294 535_TMOD_FOD_R 442 SP101_SPET11_358_38 TGGGGATTCAGCCATCAAAGCAGCTA 588SP101_SPET11_448_473_TMOD_ TCCAACCTTTTCCACAACACAATCACC 998 7_TMOD_FTTGAC R 443 SF101_SPET11_600_62 TCCTTACTTCCAACTATCAATCTTTT 348SP101_SPET11_686_714_TMOD_ TCCCATTTTTTCACGCATCCTGAAAATA 1018 9_TMOD_FGGAAC R TC 444 SP101_SPET11_658_68 TGGGGATTCATATCACCGATAAGAAG 589SP101_SPET11_756_784_TMOD TGATTGGCGATAAAGTGATATTTTCTAA 1189 4_TMOD_F AAR AA 445 SP101_SPET11_776_80 TTCGCCAATCAAAACTAAGGGAATGG 673SP101_SPET11_871_896_TMOD_ TGCCCACCAGAAACACTAGCAGGATAA 1217 1_TMOD_F C R446 SP101_SPET11_1_29_T TAACCTTAATTGGAAAGAAACCCAAG 154SP101_SPET11_92_116_TMOD_ TCCTACCCAACGTTCACCAAGGGCAG 1044 MOD_F AAGT R447 SP101_SPET11_364_38 TCACCCATCAAACCAGCTATTG 276SP101_SPET11_448_471_R TACCTTTTCCACAACAGAATCAGC 894 5_F 448SP101_SPET11_3085_3 TAGCTAATGGTCAGGCAGCC 216 SF101_SPET11_3170_3194_RTCCACGACCATCTTCCAAAGATTTC 1066 104_F 449 RPLB_EC_690_710_FTCCACACGCTCCTGGTGAACG 309 RPLB_EC_737_758_R TCTGCTCCTTTACCCCATGGAC 1336481 BONTA_X52066_538_55 TATCGCTCTACTCAA 239 BONTA_X52066_647_660_RTGTTACTGCTGGAT 1346 2_F 482 BONTA_X52066_538_55TA*TpGGC*Tp*Cp*TpA*Cp*Tp*C 143 BONTA_X52066_647_660P_RTG*Tp*TpA*Cp*TpG*Cp*TpGGAT 1146 2P_F pAA 483 BONTA_X52066_701_72GAATAGCAATTAATCCAAAT 94 BONTA_X52066_759_775_R TTACTTCTAACCCACTC 13670_F 484 BONTA_X52066_701_72 GAA*TpAG*CpAA*Tp*TpAA*Tp*C 91BONTA_X52066_759_775P_R TTA*Cp*Tp*Tp*Cp*TpAA*Cp*Cp*C 1359 p*CpAAATpA*Cp*TpC 485 BONTA_X52066_450_47 TCTAGTAATAATAGGACCCTCAGC 393BONTA_X52066_517_539_R TAACCATTTCGCGTAAGATTCAA 859 3_F 486BONTA_X52066_450_47 T*Cp*TpAGTAATAATAGGA*Cp*Cp 142BONTA_X52066_517_539P_R TAACCA*Tp*Tp*Tp*CpGCGTAAGA*T 857 3P_F*Cp*Tp*CpAGC p*Tp*CpAA 487 BONTA_X52066_591_62TGAGTCACTTGAAGTTGATACAAATC 463 BONTA_X52066_644_671_RTCATGTGCTAATGTTACTGCTGGATCTG 992 0_F CTCT 608 SSPE_BA_156_168P_FTGCTpGCpTpAGCpATT 616 SSPE_BA_243_255P_R TGCpACCpTGATpTpGT 1241 609SSPE_BA_75_89P_F TACpAGACTpTpTpGCpGAC 192 SSPE_BA_163_177P_RTGTGCTpTpTpGAATpGCpT 1338 610 SSPE_BA_150_168P_F TCCTTCTGGTpCCpTpAGCpATT533 SSPE_BA_243_2642_R TGATTGTTTTCCpAGCPTGATpTpGT 1191 611SSPE_BA_72_89P_F TCGTACpAGAGTpTPTpGCpGAC 602 SSPE_BA_163_182P_RTCATTTGTGCTpTpTpGAATpGCpT 995 612 SSPE_BA_114_1372_FTCAACCAAACGCACAATpCpAGAAGC 255 SSPE_BA_196_222P_RTTGCACGTCpTpCTTTCAGTTGCAAATT 1401 C 699 SSPE_BA_123_153_FTGCACAATCAGAAGCTAAGAAAGCGC 488 SSPE_BA_202_231_RTTTCACAGCATGCACGTCTGTTTCAGTT 1431 AAGCT GC 700 SSPE_BA_156_168_FTCGTGCTAGCATT 612 SSPE_BA_243_255_R TCCAGCTGATTCT 1202 701SSPE_BA_75_89_F TACAGACTTTGCGAC 179 SSPE_BA_163_177_R TGTGCTTTGAATGCT1338 702 SSPE_BA_150_168_F TGCTTCTCCTGCTAGCATT 533 SSPE_BA_243_264_RTGATTGTTTTGCAGCTGATTGT 1190 703 SSPE_BA_72_89_F TGGTACAGAGTTTGCGAC 600SSPE_BA_163_182_R TCATTTGTGCTTTGAATGCT 995 704 SSPE_BA_146_168_FTGCAAGCTTCTGGTGCTAGCATT 484 SSPE_BA_242_267_R TTGTGATTGTTTTGCAGCTGATTGTG1421 705 SSPE_BA_63_89_F TGCTAGTTATCCTACAGAGTTTGCGA 518SSPE_BA_163_191_R TGATAACTAGCATTTGTGCTTTGAATGC 986 C T 706SSPE_BA_114_137_F TCAAGCAAACGCACAATCAGAAGC 255 SSPE_BA_196_222_RTTGCACGTCTGTTTCAGTTGCAAATTC 1402 770 PLA_AF053945_7377_7TGACATCCGGCTCACGTTATTATGGT 442 PLA_AF053945_7434_7462_RTGTAAATTCCGCAAAGACTTTCGCATTA 1313 402_F G 771 PLA_AF053945_7382_7TCCGGCTCACGTTATTATGGTAC 327 PLA_AF053945_7482_7502_RTGGTCTGAGTACCTCCTTTGC 1304 404_F 772 PLA_AF053945_7481_7TGCAAAGGAGGTACTCAGACCAT 481 PLA_AF053945_7539_7562_RTATTGGAAATACCGGCAGCATCTC 943 503_F 773 PLA_AF053945_7186_7TTATACCGGAAACTTCCCGAAAGGAG 657 PLA_AF053945_7257_7280_RTAATGCCATACTGGCCTGCAAGTC 879 211_F 774 CAF1_AF053947_33407_TCAGTTCCGTTATCGCCATTGCAT 292 CAF1_AF053947_33494_33514_TGCGGGCTGGTTCAACAAGAG 1235 33430_F R 775 CAF1_AF053947_33515_TCACTCTTACATATAAGGAAGGCGCT 270 CAF1_AF053947_33595_33621_TCCTGTTTTATAGCCGCCAAGAGTAAG 1053 33541_F C R 776 CAF1_AF053947_33435TGGAACTATTGCAACTGCTAATG 542 CAF1_AF053947_33499_33517_TGATGCGGGCTGGTTCAAC 1183 33457_F R 777 CAF1_AF053947_33687_TCAGGATGGAAATAACCACCAATTCA 286 CAF1_AF053947_33755_33782_TCAAGGTTCTCACCGTTTACCTTAGGAG 962 33716_F CTAC R 778 INV_U22457_515_539_TGGCTCCTTGGTATGACTCTGCTTC 573 INV_U22457_571_598_RTGTTAAGTGTGTTGCGGCTGTCTTTATT 1343 F 779 INV_U22457_699_724_TGCTCAGGCCTGGACCGATTATTTAC 525 INV_U22457_753_776_RTCACGCGACGAGTGCCATCCATTG 976 F 780 INV_U22457_834_858_TTATTTACCTGCACTCCCACAACTG 664 INV_U22457_942_966_RTGACCCAAAGCTGAAAGCTTTACTG 1154 F 781 INV_U22457_1558_158TGGTAACAGAGCCTTATAGGCGCA 597 INV_U22457_1619_1643_RTTGCGTTGCAGATTATCTTTACCAA 1408 1_F 782 LL_NC003143_2366996_TGTAGCCGCTAACCACTACCATCC 627 LL_NC003143_2367073_23670TCTCATCCCGATATTACCGCCATGA 1123 2367019_F 97_R 783 LL_NC003143_2367172_TGGACCGCATCACGATTCTCTAC 550 LL_NC003143_2367249_23672TGGCAACAGCTCAACACCTTTGG 1272 2367194_F 71_R 874 RPLB_EC_649_679_FTGICCIACIGTIIGIGGTTCTGTAAT 620 RPLB_EC_739_762_TMOD_RTTCCAAGTGCTGGTTTACCCCATGG 1380 GAACC 875 RPLB_EC_642_679P_FTpCpCpTpTpGITpGTCCIACIGTII 646 RPLB_EC_739_762_TMOD_RTTCCAAGTGCTGGTTTACCCCATGG 1380 GIGGTTCTGTAATGAACC 876MECIA_Y14051_3315_3 TTACACATATCGTGAGCAATGAACTG 653MECIA_Y14051_3367_3393_R TGTGATATGGAGGTGTAGAACGTGTTA 1333 341_F A 877MECA_Y14051_3774_38 TAAAACAAACTACGGTAACATTGATC 144MECA_Y14051_3828_3854_R TCCCAATCTAACTTCCACATACCATCT 1015 02_F GCA 878MECA_Y14051_3645_36 TGAAGTAGAAATGACTGAACGTCCGA 434MECA_Y14051_3690_3719_R TGATCCTGAATGTTTATATCTTTAACGC 1181 70_F CT 879MECA_Y14051_4507_45 TCAGGTACTGCTATCCACCCTCAA 288 MECA_Y14051_4555_4581_RTGGATAGACGTCATATGAAGGTGTGCT 1269 30_F 880 MECA_Y14051_4507_45TGTACTGCTATCCACCCTCAA 626 MECA_Y14051_4586_4610_RTATTCTTCGTTACTCATGCCATACA 939 30_F 881 MECA_Y14051_4669_46TCACCAGGTTCAACTCAAAAAATATT 262 MECA_Y14051_4765_4793_RTAACCACCCCAAGATTTATCTTTTTGCC 858 98_F AACA A 882 MECA_Y14051_4520_45TCpCpACpCpCpTpCpAA 389 MECA_Y14051_4590_4600P_R TpACpTpCpATpGCpCpA 135730P_F 883 MECA_Y14051_4520_45 TCpCpACpCpCpTpCpAA 389MECA_Y14051_4600_4610P_R TpATpTpCpTpTpCpGTpT 1358 30P_F 902TRPE_AY094355_1467_ ATGTCGATTGCAATCCGTACTTGTG 36TRPE_AY094355_1569_1592_R TGCGCGAGCTTTTATTTGGGTTTC 1231 1491_F 903TRPE_AY094355_1445_ TGGATGCCATGGTGAAATGGATATGT 557TRPE_AY094355_1551_1580_R TATTTGGGTTTCATTCCACTCAGATTCT 944 1471_F C GG904 TRPE_AY094355_1278_ TCAAATGTACAAGGTGAAGTGCGTCA 247TRPE_AY094355_1392_1418_R TCCTCTTTTCACAGGCTCTACTTCATC 1048 1303_F 905TRPE_AY094355_1064_ TCGACCTTTGGCAGGAACTAGAC 357TRPE_AY094355_1171_1196_R TACATCGTTTCGCCCAAGATCAATCA 885 1086_F 906TRPE_AY094355_666_6 GTGCATGCGGATACAGAGCAGAG 135 TRPE_AY094355_769_791_RTTCAAAATGCGGAGGCGTATGTG 1372 88_F 907 TRPE_AY094355_757_7TGCAAGCGCGACCACATACG 483 TRPE_AY094355_864_883_R TGCCCAGGTACAACCTGCAT1218 76_F 908 RECA_AF251469_43_68_ TGGTACATGTGCCTTCATTGATGCTG 601RECA_AF251469_140_163_R TTCAAGTGCTTGCTCACCATTGTC 1375 F 909RECA_AF251469_169_1 TGACATGCTTGTCCGTTTCAGGC 446 RECA_AF251469_277_300_RTGGCTCATAAGACGCGCTTGTAGA 1280 90_F 910 PARC_X95819_87_110_TGGTGACTCGGCATGTTATGAAGC 609 PARC_X95819_201_2229_RTTCGGTATAACGCATCGCAGCA 1387 F 911 PARC_X95819_87_110_TGGTGACTCGGCATGTTATGAAGC 609 PARC_X95819_192_219_RGGTATAACGCATCGCAGCAAAAGATTTA 836 F 912 PARC_X95819_123_147_GGCTCAGCCATTTAGTTACCGCTAT 120 PARC_X95819_232_260_RTCGCTCAGCAATAATTCACTATAAGCCG 1081 F A 913 PARC_X95819_43_63_FTCAGCGCGTACAGTGGGTGAT 277 PARC_X95819_143_170_RTTCCCCTGACCTTCGATTAAAGGATAGC 1383 914 OMPA_AY485227_272_3TTACTCCATTATTGCTTGGTTACACT 655 OMPA_AY485227_364_388_RGAGCTGCGCCAACGAATAAATCGTC 812 01_F TTCC 915 OMPA_AY485227_379_4TGCGCAGCTCTTGGTATCGAGTT 509 OMPA_AY485227_492_519_RTGCCGTAACATAGAAGTTACCGTTGATT 1223 01_F 916 OMPA_AY485227_313_3TACACAACAATGGCGGTAAAGATGG 178 OMPA_AY485227_424_453_RTACGTCGCCTTTAACTTGGTTATATTCA 901 35_F GC 917 OMPA_AY485227_415_4TGCCTCGAAGCTGAATATAACCAAGT 506 OMPA_AY485227_514_546_RTCGGGCGTAGTTTTTAGTAATTAAATCA 1092 41_F T GAAGT 918 OMPA_AY485227_494_5TCAACGGTAACTTCTATGTTACTTCT 252 OMPA_AY485227_569_596_RTCGTCGTATTTATAGTGACCAGCACCTA 1108 20_F G 919 OMPAAY4_8522_75_515TCAAGCCGTACGTATTATTAGGTGCT 257 OMPA_AY485227_658_680_RTTTAAGCGCCAGAAAGCACCAAC 1425 77_F G 920 OMPA_AY485227_555_5TCCGTACGTATTATTAGGTGCTGGTC 328 OMPA_AY485227_635_662_RTCAACACCAGCGTTACCTAAAGTACCTT 954 81_F A 921 OMPA_AY485227_556_5TCGTACGTATTATTAGGTGCTGGTCA 379 OMPA_AY485227_659_683_RTCGTTTAAGCGCCAGAAAGCACCAA 1114 83_F CT 922 OMPA_AY485227_657_6TGTTGGTGCTTTCTGGCGCTTAA 645 OMPA_AY485227_739_765_RTAAGCCAGCAAGAGCTGTATAGTTCCA 871 79_F 923 OMPA_AY485227_660_6TGGTGCTTTCTGGCGCTTAAACGA 613 OMPA_AY485227_786_807_RTACAGGAGCAGCAGGCTTCAAG 884 83_F 924 GYRA_AF100557_4_23_TCTGCCCGTGTCGTTGGTGA 402 GYRA_AF100557_119_142_RTCGAACCGAAGTTACCCTGACCAT 1063 F 925 GYRA_AF100557_70_94_TCCATTGTTCGTATGGCTCAAGACT 316 GYRA_AF100557_178_201_RTGCCAGCTTAGTCATACGGACTTC 1211 F 926 GYRB_AB008700_19_40_TCAGGTGGCTTACACGGCGTAG 289 GYRB_AB008700_111_140_RTATTGCGGATCACCATGATGATATTCTT 941 F GC 927 GYRB_AB008700_265_2TCTTTCTTGAATGCTGGTGTACGTAT 420 GYRB_AB008700_369_395_RTCGTTGAGATGGTTTTTACCTTCGTTG 1113 92_F CG 928 GYRB_AB008700_368_3TCAACGAAGGTAAAAACCATCTCAAC 641 GYRB_AB008700_466_494_RTTTGTGAAACAGCGAACATTTTCTTGGT 1440 94_F G A 929 GYRB_AB008700_477_5TGTTCGCTGTTTCACAAACAACATTC 641 GYRB_AB008700_611_632_RTCACGCGCATCATCACCAGTCA 977 04_F CA 930 GYRB_AB008700_760_7TACTTACTTGAGAATCCACAAGCTGC 198 GYRB_AB008700_862_888_RACCTGCAATATCTAATGCACTCTTACG 729 87_F AA 931 WAAA_Z96925_2_29_FTCTTGCTCTTTCGTGAGTTCAGTAAA 416 WAAA_Z96925_115_138_RCAAGCGGTTTGCCTCAAATAGTCA 758 TG 932 WAAA_Z96925_286_311_TCGATCTGGTTTCATGCTGTTTCAGT 360 WAAA_Z96925_394_412_R TGGCACGAGCCTGACCTGT1274 F 939 RPOB_EC_3798_3821_F TGGGCAGCGTTTCGGCGAAATGGA 581RPOB_EC_3862_3889_R TGTCCGACTTGACGGTCAGCATTTCCTG 1326 940RPOB_EC_3798_3821_F TGGGCAGCGTTTCGGCGAAATGGA 581 RPOB_EC_3862_3889_2_RTGTCCGACTTGACGGTTAGCATTTCCTG 1327 941 TUFB_EC_272_299_FTGATCACTGGTGCTGCTCAGATGGA 468 TUFB_EC_337_362_RTGGATGTGCTCACGAGTCTGTGGCAT 1271 942 TUFB_EC_251_278_FTGCACGCCGACTATGTTAAGAACATG 493 TUFB_EC_337_360_RTATGTGCTCACGAGTTTGCGGCAT 937 AT 949 GYRB_AB008700_760_7TACTTACTTGAGAATCCACAAGCTGC 198 GYRB_AB008700_862_888_2_RTCCTGCAATATCTAATGCACTCTTACG 1050 87_F AA 958 RPOC_EC_2223_2243_FTGGTATGCGTGGTCTGATGGC 605 RPOC_EC_2329_2352_R TGCTAGACCTTTACGTGCACCGTG1243 959 RPOC_EC_918_938_F TCTGGATAACGGTCGTCGCGG 404 RPOC_EC_1009_1031_RTCCAGCAGGTTCTGACGGAAACG 1004 960 RPOC_EC_2334_2357_FTGCTCGTAAGGGTCTGGCGGATAC 523 RPOC_EC_2380_2403_RTACTAGACGACGGGTCAGGTAACC 905 961 RPOC_EC_917_938_FTATTGGACAACCGTCGTCGCGG 242 RPOC_EC_1009_1034_RTTACCGAGCACGTTCTGACGGAAACG 1362 962 RPOB_EC_2005_2027_FTCGTTCCTGCAACACGATGACGC 387 RPOB_EC_2041_2064_R TTGACGTTGCATGTTCGAGCCCAT1399 963 RPOB_EC_1527_1549_F TCAGCTGTCGCAGTTCATGGACC 282RPOB_EC_1630_1649_R TCGTCGCGGACTTCGAAGCC 1104 964 INFB_EC_1347_1367_FTGCGTTTACCOCAATGCGTGC 515 INFB_EC_1414_1432_R TCGGCATCACGCCGTCGTC 1090965 VALS_EC_1128_1151_F TATCCTGACCGACCAGTGCTACGT 237 VALS_EC_1231_1257_RTTCGCGCATCCAGGAGAACTACATGTT 1384 978 RPOC_EC_2145_2175_FTCAGGAGTCGTTCAACTCGATCTACA 285 RPOC_EC_2228_2247_R TTACGCCATCACGCCACGCA1363 TGATG 1045 CJST_CJ_1668_1700_F TGCTCGAGTGATTGACTTTGCTAAAT 522CJST_CJ_1774_1799_R TGACCGTGTGGAAAAGGACTTCGATG 1170 TTAGAGA 1046CJST_CJ_2171_2197_F TCCTTTGGTGGTCGTAGATGAAAAAG 388 CJST_CJ_2283_2313_RTCTCTTTCAAAGCACCATTGCTCATTAT 1126 G AGT 1047 CJST_CJ_584_616_FTCCAGGACAAATGTATGAAAAATGTC 315 CJST_CJ_663_692_RTTCATTTTCTGGTCCAAAGTAAGCAGTA 1379 CAAGAAG TC 1048 CJST_CJ_360_394_FTCCTGTTATCCCTGAAGTAGTTAATC 346 CJST_CJ_442_476_RTCAACTGGTTCAAAAACATTAAGTTGTA 955 AAGTTTGTT ATTGTCC 1049CJST_CJ_2636_2668_F TGCCTAGAACATCTTAAAAATTTCCG 504 CJST_CJ_2753_2777_RTTGCTGCCATAGCAAAGCCTACACC 1409 CCAACTT 1050 CJST_CJ_1290_1320_FTGGCTTATCCAAATTTAGATCGTGGT 575 CJST_CJ_1406_1433_RTTTCCTCATGATCTGCATGAAGCATAAA 1437 TTTAC 1051 CJST_CJ_3267_3293_FTTTCATTTTACGCCCTCCTCCAGGTC 707 CJST_CJ_3356_3385_RTCAAAGAACCCGCACCTAATTCATCATT 951 G TA 1052 CJST_CJ_5_39_FTAGGCGAAGATATACAAAGAGTATTA 222 CJST_CJ_104_137_RTCCCTTATTTTTCTTTCTACTACCTTCG 1029 GAAGCTAGA GATAAT 1053CJST_CJ_1080_1110_F TTGAGGGTATGCACCGTCTTTTTGAT 681 CJST_CJ_1166_1198_RTCCCCTCATGTTTAAATGATCAGGATAA 1022 TCTTT AAAGC 1054 CJST_CJ_2060_2090_FTCCCGGACTTAATATCAATGAAAATT 323 CJST_CJ_2148_2174_RTCGATCCGCATCACCATCAAAAGCAAA 1068 GTGGA 1055 CJST_CJ_2869_2895_FTGAAGCTTGTTCTTTAGCAGGACTTC 432 CJST_CJ_2979_3007_RTCCTCCTTGTGCCTCAAAACGCATTTTT 1045 A A 1056 CJST_CJ_1880_1910_FTCCCAATTAATTCTGCCATTTTTCCA 317 CJST_CJ_1981_2011_RTGGTTCTTACTTGCTTTGCATAAACTTT 1309 GGTAT CCA 1057 CJST_CJ_2185_2212_FTAGATGAAAAGGGCGAAGTGGCTAAT 208 CJST_CJ_2283_2316_RTGAATTCTTTCAAAGCACCATTGCTCAT 1152 GG TATAGT 1058 CJST_CJ_1643_1670_FTTATCGTTTGTGGAGCTAGTGCTTAT 660 CJST_CJ_1724_1752_RTGCAATGTGTGCTATGTCAGCAAAAAGA 1198 GC T 1059 CJST_CJ_2165_2194_FTGCGGATCGTTTGGTGGTTGTAGATG 511 CJST_CJ_2247_2278_RTCCACACTGGATTGTAATTTACCTTGTT 1002 AAAA CTTT 1060 CJST_CJ_599_632_FTGAAAAATGTCCAAGAAGCATAGCAA 424 CJST_CJ_711_743_RTCCCGAACAATGAGTTGTATCAACTATT 1024 AAAAAGCA TTTAC 1061 CJST_CJ_360_393_FTCCTGTTATCCCTGAAGTAGTTAATC 345 CJST_CJ_443_477_RTACAACTGGTTCAAAAACATTAAGCTGT 882 AAGTTTGT AATTGTC 1062CJST_CJ_2678_2703_F TCCCCAGGACACCCTGAAATTTCAAC 321 CJST_CJ_2760_2787_RTGTGCTTTTTTTGCTGCCATAGCAAAGC 1339 1063 CJST_CJ_1268_1299_FAGTTATAAACACGGCTTTCCTATGGC 29 CJST_CJ_1349_1379_RTCGGTTTAAGCTCTACATGATCGTAAGG 1096 TTATCC ATA 1064 CJST_CJ_1650_1713_FTGATTTTGCTAAATTTAGACAAATTG 479 CJST_CJ_1795_1822_RTATGTGTAGTTGAGCTTACTACATGAGC 938 CGGATGAA 1065 CJST_CJ_2857_2887_FTGGCATTTCTTATGAAGCTTGTTCTT 565 CJST_CJ_2965_2998_RTGCTTCAAAACGCATTTTTACATTTTCG 1253 TAGCA TTAAAG 1070 RNASEP_BKM_580_599_TGCGGGTAGGGAGCTTGAGC 512 RNASEP_BKM_665_686_R TCCGATAAGCCGGATTCTGTGC1034 F 1071 RNASEP_BKM_616_637_ TCCTAGAGGAATGGCTGCCACG 333RNASEP_BKM_665_687_R TGCCGATAAGCCGGATTCTGTGC 1222 F 1072RNASEP_BKM_574_592_ TGGCACGGCCATCTCCGTG 561 RNASEP_BKM_616_635_RTCGTTTCACCCTGTCATGCCG 1115 F 1073 23S_BRM_1110_1129_FTGCGCGGAAGATGTAACGGG 510 23S_BRM_1176_1201_R TCGCAGGCTTACAGAACGCTCTCCTA1074 1074 23S_BRM_515_536_F TGCATACAAACAGTCGGAGCCT 496 23S_BRM_616_635_RTCGGACTCGCTTTCGCTACG 1088 1075 RNASEP_CLB_459_487_TAAGGATAGTGCAACAGAGATATACC 162 RNASEP_CLB_498_526_RTGCTCTAACCTCACCGTTCCACCCTTAC 1247 F GCC C 1076 RNASEP_CLB_459_487_TAAGGATAGTGCAACAGAGATATACC 162 RNASEP_CLB_498_522_RTTTACCTCGCCTTACCACCCTTACC 1426 F GCC 1077 ICD_CXB_93_120_FTCCTGACCGACCCATTATTCCCTTTA 343 ICD_CXB_172_194_R TAGGATTTTTCCACGGCGGCATC921 TC 1078 ICD_CXB_92_120_F TTCCTGACCGACCCATTATTCCCTTT 671 ICDCXB_172_194_R TAGGATTTTTCCACGGCGGCATC 921 ATC 1079 ICD_CXB_176_198_FTCGCCGTGGAAAAATCCTACGCT 369 ICD_CXB_224_247_R TAGCCTTTTCTCCGGCGTAGATCT916 1080 IS1111A_NC002971_74 TCAGTATGTATCCACCGTAGCCAGTC 290IS1111A_NC002971_6928_695 TAAACGTCCGATACCAATGGTTCGCTC 848 66_6891_F 4_R1081 IS1111A_NC002971_74 TGGGTGACATTCATCAATTTCATCGT 594IS1111A_NC002971_7529_755 TCAACAACACCTCCTTATTCCCACTC 952 56_7483_F 4_R1082 RNASEP_RKP_419_448_ TGGTAAGAGCGCACCGGTAAGTTGGT 599RNASEP_RKP_542_565_R TCAAGCGATCTACCCGCATTACAA 957 F AACA 1083RNASEP_RKP_422_443_ TAAGAGCGCACCGGTAAGTTSG 159 RNSASEP_RKP_542_565_RTCAAGCGATCTACCCGCATTACAA 957 F 1084 RNASEP_RKP_466_491_TCCACCAAGAGCAAGATCAAATAGGC 310 RNASEP_RKP_542_565_RTCAAGCGATCTACCCGCATTACAA 957 F 1085 RNASEP_RKP_264_287_TCTAAATGGTCGTGCAGTTGCGTG 391 RNASEP_RKP_295_321_RTCTATAGAGTCCGGACTTTCCTCGTGA 1119 F 1086 RNASEP_RKP_426_448_TGCATACCGGTAAGTTGGCAACA 497 RNASEP_RKP_542_565_RTCAAGCGATCTACCCGCATTACAA 957 F 1087 OMPB_RKP_860_890_FTTACAGGAAGTTTAGGTGGTAATCTA 654 OMPB_RKP_972_996_RTCCTGCAGCTCTACCTGCTCCATTA 1051 AAAGG 1088 OMPB_RKP_1192_1221_TCTACTGATTTTGGTAATCTTGCAGC 392 OMPH_RKP_1288_1315_RTAGCAgCAAAAGTTATCACACCTGCAGT 910 F ACAG 1089 OMPB_RKP_3417_3440_TGCAAGTGGTACTTCAACATGGGG 485 OMPH_RKP_3520_3550_RTGGTTGTAGTTCCTGTAOTTGTTGCATT 1310 F AAC 1090 GLTA_RKP_1043_1072_TGGGACTTGAAGCTATCGCTCTTAAA 576 GLTA_RKP_1138_1162_RTGAACATTTGCGACGGTATACCCAT 1147 G GATG 1091 GLTA_RKP_400_428_FTCTTCTCATCCTATGGCTATTATGCT 413 GLTA_RKP_499_529_RTGGTGGGTATCTTAGCAATCATTCTAAT 1305 TGC AGC 1092 GLTA_RKP_1023_1055_TCCGTTCTTACAAATAGCAATAGAAC 330 GLTA_RKP_1129_1156_RTTGGCGACGGTATACCCATAGCTTTATA 1415 F TTGAAGC 1093 GLTA_RKP_1043_1072_TGGAGCTTGAAGCTATCGCTCTTAAA 553 GLTA_RKP_1138_1162_RTGAACATTTGCGACGGTATACCCAT 1147 2_F GATG 1094 GLTA_RKP_1043_1072_TGGAACTTGAAGCTCTCGCTCTTAAA 543 GLTA_RKP_1138_1164_RTGTGAACATTTGCGACGGTATACCCAT 1330 3_F GATG 1095 GLTA_RKP_400_428_FTCTTCTCATCCTATGGCTATTATGCT 413 GLTA_RKP_505_534_RTGCGATGGTAGGTATCTTAGCAATCATT 1230 TGC CT 1096 CTXA_VBC_117_142_FTCTTATGCCAAGAGGACAGAGTGAGT 410 CTXA_VBC_194_218_RTGCCTAACAAATCCCGTCTGAGTTC 1226 1097 CTXA_VBC_351_377_FTGTATTAGGGGCATACAGTCCTCATC 630 CTXA_VBC_441_466_RTGTCATCAAGCACCCCAAAATGAACT 1324 C 1098 RNASEP_VBC_331_349_TCCGCGGAGTTGACTGGGT 325 RNASEP_VBC_388_414_R TGACTTTCCTCCCCCTTATCAGTCTCC1163 F 1099 TOXR_VBC_135_158_F TCGATTAGGCAGCAACGAAAGCCG 362TOXR_VBC_221_246_R TTCAAAACCTTGCTCTCGCCAAACAA 1370 1100 ASD_FRT_1_29_FTTGCTTAAAGTTGGTTTTATTGGTTG 690 ASD_FRT_86_116_RTGAGATGTCGAAAAAAACGTTGGCAAAA 1164 GCG TAC 1101 ASD_FRT_43_76_FTCAGTTTTAATGTCTCGTATGATCGA 295 ASD_FRT_129_156_RTCCATATTGTTGCATAAAACCTGTTGGC 1009 ATCAAAAG 1102 GALE_FRT_165_199_FTTATCAGCTAGACCTTTTAGGTAAAG 658 GALE_FRT_241_269_RTCACCTACAGCTTTAAAGCCAGCAAAAT 973 CTAAGC G 1103 GALE_FRT_834_865_FTCAAAAAGCCCTAGGTAAAGAGATTC 245 GALE_FRT_901_925_RTAGCCTTGGCAACATCAGCAAAACT 915 CATATC 1104 GALE_FRT_308_339_FTCCAAGGTACACTAAACTTACTTGAG 306 GALE_FRT_390_422_RTCTTCTGTAAAGGGTGGTTTATTATTCA 1136 CTAATG TCCCA 1105 IPAH_SGF_258_277_FTGAGGACCGTGTCGCGCTCA 458 IPAH_SGF_301_327_R TCCTTCTGATGCCTGATGGACCAGGAG1055 1106 IPAH_SGF_113_134_F TCCTTGACCGCCTTTCCGATAC 350IPAH_SGF_172_191_R TTTTCCAGCCATGCAGCGAC 1441 1107 IPAH_SGF_462_486_FTCAGACCATGCTCGCAGAGAAACTT 271 IPAH_SGF_522_540_R TGTCACTCCCGACACGCCA1322 1111 RNASEP_BRM_461_488_ TAAACCCCATCGGGAGCAAGACCGAA 147RNASEP_BRM_542_561_R TGCCTCGCGCAACCTACCCG 1227 F TA 1112RNASEP_BRM_325_347_ TACCCCAGGGAAAGTGCCACAGA 185 RNASEP_BRM_402_428_RTCTCTTACCCCACCCTTTCACCCTTAC 1125 F 1128 HUPB_CJ_113_134_FTAGTTGCTCAAACAGCTGGGCT 230 HUPB_CJ_157_188_RTCCCTAATAGTAGAAATAACTGCATCAG 1028 TAGC 1129 HUPB_CJ_76_102_FTCCCGGAGCTTTTATGACTAAAGCAG 324 HUPB_CJ_157_188_RTCCCTAATAGTAGAAATAACTGCATCAG 1028 AT TAGC 1130 HUPB_CJ_76_102_FTCCCGGAGCTTTTATGACTAAAGCAG 324 HUPB_CJ_114_135_R TAGCCCAGCTGTTTGAGCAACT913 AT 1151 AB_MLST-11- TGAGATTGCTGAACATTTAATGCTGA 454 AB_MLST-11-TTGTACATTTGAAACAATATGCATGACA 1418 OIF007_62_91_F TTGA OIF007_169_203_RTGTGAAT 1152 AB_MLST-11- TATTGTTTCAAATGTACAAGGTGAAG 243 AB_MLST-11-TCACAGGTTCTACTTCATCAATAATTTC 969 OIF007_185_214_F TGCG OIF007_291_324_RCATTGC 1153 AB_MLST-11- TGGAACGTTATCAGGTGCCCCAAAAA 541 AB_MLST-11-TTGCAATCGACATATCCATTTCACCATG 1400 OIF007_260_289_F TTCG OIF007_364_393_RCC 1154 AB_MLST-11- TGAAGTGCGTGATGATATCGATGCAC 436 AB_MLST-11-TCCGCCAAAAACTCCCCTTTTCACAGG 1036 OIF007_206_239_F TTGATGTAOIF007_318_344_R 1155 AB_MLST-11- TCGGTTTAGTAAAAGAACGTATTGCT 378AB_MLST-11- TTCTGCTTGAGGAATAGTGCGTGG 1392 OIF007_522_552_F CAACCOIF007_587_610_R 1156 AB_MLST-11- CCAACCTGACTGCGTGAATGGTTGT 250AB_MLST-11- TACGTTCTACGATTTCTTCATCAGGTAC 902 OIF007_547_571_FOIF007_656_68_R ATC 1157 AB_MLST-11- TCAAGCAGAAGCTTTGGAAGAAGAAG 256AB_MLST-11- TACAACGTGATAAACACGACCAGAAGC 881 OIF007_601_627_F GOIF007_710_736_R 1158 AB_MLST-11- TCGTGCCCGCAATTTGCATAAAGC 384AB_MLST-11- TAATGCCGGGTAGTGCAATCCATTCTTC 878 OIF007_1202_1225_FOIF007_1266_1296_R TAG 1159 AB_MLST-11- TCGTGCCCGCAATTTGCATAAAGC 384AB_MLST-11- TGCACCTGCGGTCGAGCG 1199 OIF007_1202_1225_FOIF007_1299_1316_R 1160 AB_MLST-11- TTGTAGCACAGCAAGGCAAATTTCCT 694AB_MLST-11- TGCCATCCATAATCACGCCATACTGACG 1215 OIF007_1234_1264_FOIF007_1335_1362_R 1161 AB_MLST-11- TAGGTTTACGTCAGTATGGCGTGATT 225AB_MLST-11- TGCCAGTTTCCACATTTCACGTTCGTG 1212 OIF007_1327_1356_FOIF007_1422_1448_R 1162 AB_MLST-11- TCGTGATTATGGATGGCAACGTGAA 383AB_MLST-11- TCGCTTGAGTGTAGTCATGATTGCG 1083 OIF007_1345_1369_FOIF007_1470_1494_R 1163 AB_MLST-11- TTATGGATGGCAACGTGAAACGCGT 662AB_MLST-11- TCGCTTGAGTGTAGTCATGATTGCG 1083 OIF007 1351_1375_FOIF007_1470_1494_R 1164 AB_MLST-11- TCTTTGCCATTGAAGATGACTTAAGC 422AB_MLST-11- TCGCTTGAGTGTAGTCATGATTGCG 1083 OIF007_1387_1412_FOIF007_1470_1494_R 1165 AB_MLST-11- TACTAGCGGTAAGCTTAAACAAGATT 194AB_MLST-11- TGAGTCGGGTTCACTTTACCTGGCA 1173 OIF007_1542_1569_F GCOIF007_1656_1680_R 1166 AB_MLST-11- TTGCCAATGATATTCGTTGGTTAGCA 684AB_MLST-11- TGAGTCGGGTTCACTTTACCTGGCA 1173 OIF007_1566_1593_F AGOIF007_1656_1650_R 1167 AB_MLST-11- TCGGCGAAATCCGTATTCCTGAAAAT 375AB_MLST-11- TACCGGAAGCACCAGCGACATTAATAG 890 OIF007_1611_1638_F GAOIF007_1731_1757_R 1168 AB_MLST-11- TACCACTATTAATGTCGCTGGTGCTT 182AB_MLST-11-T GCAACTGAATAGATTGCAGTAAGTTAT 1195 OIF007_1726_1752_F COIF007_1790_1821_R AAGC 1169 AB_MLST-11- TTATAACTTACTGCAATCTATTCAGT 656AB_MLST-11- TGAATTATGCAAGAAGTGATCAATTTTC 1151 OIF007_1792_1826_FTGCTTGGTG OIF007_1876_1909_R TCACGA 1170 AB_MLST-11-TTATAACTTACTGCAATCTATTCAGT 656 AB_MLST-11- TGCCGTAACTAACATAAGAGAATTATGC1224 OIF007_1792_1826_F TGCTTGGTG OIF007_1895_1927_R AAGAA 1171AB_MLST-11- TGGTTATGTACCAAATACTTTGTCTG 618 AB_MLST-11-TGACGGCATCGATACCACCGTC 1157 OIF007_1970_2002_F AAGATGGOIF007_2097_2118_R 1172 RNASEP_BRM_461_488_ TAAACCCCATCGGGAGCAAGACCGAA147 RNASEP_BRM_542_561_2_R TGCCTCGTGCAACCCACCCG 1228 F TA 2000CTXB_NC002505_46_70_ TCAGCGTATGCACATGGAACTCCTC 278CTXB_NC002505_132_162_R TCCGGCTAGAGATTCTGTATACGACAAT 1039 F ATC 2001FUR_NC002505_87_113_ TGAGTGCCAACATATCAGTGCTGAAG 465FUR_NC002505_205_228_R TCCGCCTTCAAAATGGTGGCGAGT 1037 F A 2002FUR_NC002505_87_113_ TGAGTGCCAACATATCAGTGCTGAAG 465FUR_NC002505_178_205_R TCACGATACCTGCATCATCAAATTGGTT 974 F A 2003GAPA_NC002505_533_5 TCGACAACACCATTATCTATGGTGTG 356GAPA_NC002505_646_671_R TCAGAATCGATGCCAAATGCGTCATC 980 60_F AA 2004GAPA_NC002505_694_7 TCAATGAACGACCAACAAGTGATTGA 259GAPA_NC002505_769_798_R TCCTCTATGCAACTTAGTATCARCAGGA 1046 21_F TG AT2005 GAPA_NC002505_753_7 TGCTAGTCAATCTATCATTCCGGTTG 517GAPA_NC002505_856_881_R TCCATCGCAGTCACGTTTACTGTTGG 1011 82_F ATAC 2006GYRB_NC002505_2_32_ TGCCGGACAATTACGATTCATCGAGT 501GYRB_NC002505_109_134_R TCCACCACCTCAAAGACCATGTGGTG 1003 F ATTAA 2007GYRB_NC002505_123_1 TGAGGTGGTGGATAACTCAATTGATG 460GYRB_NC002505_199_225_R TCCGTCATCGCTGACAGAAACTGAGTT 1042 52_F AAGC 2008GYRB_NC002505_768_7 TATGCAGTGGAACGATGGTTTCCAAG 236GYRB_NC002505_832_860_R TGGAAACCGGCTAAGTGAGTACCACCAT 1262 94_F A C 2009GYRB_NC002505_837_8 TGGTACTCACTTAGCGGGTTTCCG 603 GYRS_NC002505_937_957_RTCCTTCACGCGCATCATCACC 1054 60_F 2010 GYRB_NC002505_934_9TCGGGTGATGATGCGCGTGAAGG 377 GYRB_NC002505_982_1007_RTGGCTTGAGAATTTAGGATCCGGCAC 1283 56_F 2011 GYRB_NC002505_1161_TAAAGCCCGTGAAATGACTCGTCGTA 148 GYRB_NC002505_1255_1284_RTGAGTCACCCTCCACAATGTATAGTTCA 1172 1190_F AAGG GA 2012OMPU_NC002505_85_11 TACGCTGACGGAATCAACCAAAGCGG 190OMPU_NC002505_154_180_R TGCTTCAGCACGGCCACCAACTTCTAG 1254 0_F 2013OMPU_NC002505_258_2 TGACGGCCTATACGGTGTTGGTTTCT 451OMPU_NC002505_346_369_R TCCGAGACCAGCGTAGGTGTAACG 1033 83_F 2014OMPU_NC002505_431_4 TCACCGATATCATGGCTAACCACGG 266OMFU_NC002505_544_567_R TCGGTCAGCAAAACGGTAGCTTGC 1094 55_F 2015OMPU_NC002505_533_5 TAGGCGTGAAAGCAAGCTACCGTTT 223OMPU_NC002505_625_651_R TAGAGAGTAGCCATCTTCACCGTTGTC 908 57_F 2016OMPU_NC002505_689_7 TAGGTGCTGGTTACGCAGATCAAGA 224OMPU_NC002505_725_751_R TGGGGTAAGACGCGGCTAGCATGTATT 1291 13_F 2017OMPU_NC002505_727_7 TACATGCTAGCCGCGTCTTAC 181 OMPU_NC002505_811_835_RTAGCAGCTAGCTCGTAACCAGTGTA 911 47_F 2018 OMPU_NC002505_931_9TACTACTTCAAGCCGAACTTCCG 193 OMPU_NC002505_1033_1053_RTTAGAAGTCGTAACGTGGACC 1368 53_F 2019 OMPU_NC002505_927_9TACTTACTACTTCAAGCCGAACTTCC 197 OMPU_NC002505_1033_1054_RTGGTTAGAAGTCGTAACGTGGACC 1307 53_F 2020 TCPA_NC002505_48_73_TCACGATAAGAAAACCGGTCAAGAGG 269 TCPA_NC002505_148_170_RTTCTGCGAATCAATCGCACGCTG 1391 F 2021 TDH_NC004605_265_28TGGCTGACATCCTACATGACTGTGA 574 TDH_NC004605_357_386_RTGTTGAAGCTGTACTTGACCTGATTTTA 1351 9_F CG 2022 VVHA_NC004460_772_8TCTTATTCCAACTTCAAACCGAACTA 412 VVHA_NC00446O_862_886_RTACCAAAGCGTGCACGATAGTTGAG 887 02_F TGACG 2023 23S_EC_2643_2667_FTGCCTGTTCTTAGTACGAGAGGACC 508 23S_EC_2746_2770_RTGGGTTTCGCGCTTAGATGCTTTCA 1297 2024 16S_EC_713_732_TMOD_TAGAACACCGATGGCGAAGGC 202 16S_EC_789_811_R TGCGTGGACTACCAGGGTATCTA 1240F 2025 16S_EC_784_806_F TGGATTAGAGACCCTGGTAGTCC 56016S_EC_880_897_TMOD_R TGGCCGTACTCCCCAGGCG 1278 2026 16S_EC_959_981_FTGTCGATGCAACGCGAAGAACCT 634 16S_EC_1052_1074_R TACGAGCTGACGACAGCCATGCA896 2027 TUFB_EC_956_979_F TGCACACGCCGTTCTTCAACAACT 489TUFB_EC_1034_1058_2_R TGCATCACCATTTCCTTGTCCTTCG 1204 2028RPOC_EC_2146_2174_T TCAGGAGTCGTTCAACTCGATCTACA 284 RPOCEC_2227_2249_RTGCTAGGCCATCAGGCCACGCAT 1244 MOD_F TGAT 2029 RPOB_EC_1841_1866_FTGGTTATCGCTCAGGCGAACTCCAAC 617 RPOB_EC_1909_1929_TMOD_RTGCTGGATTCGCCTTTGCTACG 1250 2030 RPLB_EC_650_679_TMOTGACCTACAGTAAGAGGTTCTGTAAT 449 RPLB_EC_739_763_RTGCCAAGTGCTGGTTTACCCCATGG 1208 D_F GAACC 2031 RFLB_EC_690_710_FTCCACACGGTGGTGGTGAAGG 309 RPLB_BC_737_760_R TGGGTGCTGGTTTACCCCATGGAG1295 2032 INFB_EC_1366_1393_F TCTCGTGGTGCACAAGTAACGGATAT 397INFB_BC_1439_1469_R TGTGCTGCTTTCGCATGGTTAATTGCTT 1335 TA CAA 2033VALS_EC_1105_1124_T TCGTCCCGGCGTGGTTATCGA 385 VALS_EC_1195_1219_RTGGGTACGAACTGGATGTCGCCGTT 1292 MOD_F 2034 SSPE_BA_113_137_FTGCAACCAAACGCACAATCAGAAGC 482 SSPE_BA_197_222_TMOD_RTTGCACGTCTGTTTCAGTTGCAAATTC 1402 2035 RPOC_EC_2218_2241_TTCTGGCAGGTATGCGTCGTCTGATG 405 RPOC_EC_2313_2338_RTGGCACCGTGGGTTGAGATGAAGTAC 1273 MOD_F 2056 MECI-R_NC003923-TTTACACATATCGTGACCAATGAACT 698 MECI-R_NC003923-41798-TTGTGATATGGAGGTGTAGAAGGTGTTA 1420 41798-41609_33_60_F GA 41609_86_113_R2057 AGR-III_NC003923- TCACCAGTTTGCCACGTATCTTCAA 263AGT-III_NC003923-2108074- ACCTGCATCCCTAAACGTACTTGC 730 21080742109507_56_79_R 2109507_1_23_F 2058 AGR-III_NC003923-TGAGCTTTTAGTTGACTTTTTCAACA 457 AGR-III_NC003923-2108074-TACTTCAGCTTCGTCCAATAAAAAATCA 906 2108074- GC 2109507_622_653_R CAAT2109507569_596_F 2059 AGR-III_NC003923- TTTCACACAGCGTGTTTATAGTTCTA 701AGR-III_NC003923-2108074- TGTAGGCAAGTGCATAAGAAATTGATAC 1319 2108074- CCA2109507_1070_1098_R A 21095071024_1052_F 2060 AGR-TGGTCACTTCATAATGGATGAAGTTG 610 AGR-I_AJ617706_694_726_RTCCCCATTTAATAATTCCACCTACTATC 1021 I_AJ617706_622_651_ AAGT ACACT F 2061AGR- TGGGATTTTAAAAAACATTGGTAACA 579 AGR-I_AJ617706_626_655_RTGGTACTTCAACTTCATCCATTATGAAG 1302 L_AJ617706_580_611- TCGCAG TC F 2062AGR-II_NC002745- TCTTGCAGCAGTTTATTTGATGAACC 415 AGR-II_NC002745-2079448-TTGTTTATTGTTTCCATATGCTACACAC 1424 2079448- TAAAGT 2080879_700_731_R TTTC2080879_620_651_F 2063 AGR-II_NC002745- TGTACCCGCTGAATTAACGAATTTAT 624AGR-II_NC002745-2079448- TCGCCATAGCTAAGTTGTTTATTGTTTC 1077 2079448-ACGAC 2080879_715_745_R CAT 2080879_649_679_F 2064 AGR-TGGTATTCTATTTTGCTGATAATGAC 606 AGR- TGCCCTATCAACGATTTTGACAATATAT 1233IV_AJ617711_931_961_ CTCGC IV_AJ617711_1004_1035_R GTGA F 2065 AGR-TGGCACTCTTGCCTTTAATATTAGTA 562 AGR-IV_AJ617711_309_335_RTCCCATACCTATGGCGATAACTGTCAT 1017 IV_AJ617711_250_283_ AACTATCA F 2066BLAZ_NC002952(191382 TCCACTTATCGCAAATGGAAAATTAA 312BLAZ_18C002952(1913827 . . . TGGCCACTTTTATCAGCAACCTTACAGT 1277 7 . . .1914672)_68_68_ GCAA 1914672)_68_68_R C F 2067 BLAZ_NC002952(191382TGCACTTATCOCAAATGGAAAATTAA 494 BLAZ_NC002952(1913827 . . .TAGTCTTTTGGAACACCGTCTTTAATTA 926 7 . . . 1914672)_68_68_ GCAA1914672)68_68_2_R AAGT 2_F 2068 BLAZ_NC002952(191382TGATACTTCAACGCCTGCTGCTTTC 467 BLAZ_NC002952(1913827 . . .TGGAACACCGTCTTTAATTAAAGTATCT 1263 7 . . . 1914672)_68_68_1914672)_68_68_3_R CC 3_F 2069 BLAZ_NC002952(191382TATACTTCAACGCCTGCTGCTTTC 232 BLAZ_NC002952(1913827 . . .TCTTTTCTTTGCTTAATTTTCCATTTGC 1145 7 . . . 1914672)68681914672)_68_68_4_R GAT 4_F 2070 BLAZ_NC002952(191382TGCAATTGCTTTAGTTTTAAGTGCAT 487 BLAZ_NC002952(1913827 . . .TTACTTCCTTACCACTTTTAGTATCTAA 1366 7 . . . 1914672)_1_33_F GTAATTC1914672)_34_67_R ACCATA 2071 BLAZ_NC002952(191382TCCTTGCTTTAGTTTTAAGTGCATGT 351 BLAZ_NC002952(1913827 . . .TGGGGACTTCCTTACCACTTTTAGTATC 1289 7 . . . 1914672)_3_34_F AATTCAA1914672)_40_68_R TAA 2072 BSA-A_NC003923- TAGCGAATGTGGCTTTACTTCACAAT 214BSA-A_NC003923-1304065- TGCAAGGGAAACCTAGAATTACAAACCC 1197 1304065- T1303589_165_193_R T 1303589_99_125_F 2073 BSA-A_NC003923-ATCAATTTGGTGGCCAAGAACCTGG 32 BSA-A_NC003923-1304065-TGCATAGGGAAGGTAACACCATAGTT 1203 1304065- 1303589_253_278_R1303589_194_218_F 2074 BSA-A_NC003923- TTGACTGCGGCACAACACGGAT 679BSA-A_NC003923-1304065- TAACAACGTTACCTTCGCGATCCACTAA 856 1304065-130358_388_415_R 1303589_328_349_F 2075 BSA-ANC003923-TGCTATGGTGTTACCTTCCCTATGCA 519 BSA-A_NC003923-1304065-TGTTGTGCCGCAGTCAAATATCTAAATA 1353 1304065- 1303589_317_344_R1303589_253_278_F 2076 BSA-B_NC003923- TAGCAACAAATATATCTGAAGCAGCG 209BSA-B_NC003923-1917149- TGTGAAGAACTTTCAAATCTGTGAATCC 1331 1917149- TACT1914156_1011_1039_R A 1914156_953_982_F 2077 BSA-B_NC003923-TGAAAAGTATGGATTTGAACAACTCG 426 NSA-B_NC003923-1917149-TCTTCTTGAAAAATTGTTGTCCCGAAAC 1138 1917149- TGAATA 1914156_1109_1136_R1914156_1050_1081_F 2078 NSA-B_NC003923- TCATTATCATGCGCCAATGAGTGCAG 300NSA-B_NC003923-1917149- TGGACTAATAACAATGAGCTCATTGTAC 1267 1917149- A1914156_1323_1353_R TGA 1914156_1260_1286_F 2079 NSA-BNC003923-TTTCATCTTATCGAGGACCCGAAATC 703 NSA-B_NC003923-1917149-TGAATATGTAATGCAAACCAGTCTTTGT 1148 1917149- GA 1914156_2186_2216_R CAT1914156_2126_2153_F 2080 ERMA_NC002952- TCGCTATCTTATCGTTGAGAAGGGAT 372ERMA_NC002952-55890- TGAGTCTACACTTGGCTTAGGATGAAA 1174 55890- T56621_487_513_R 56621_366_392_F 2081 ERMA_NC002952-TAGCTATCTTATCGTTGAGAAGGGAT 217 ERMA_NC002952-55890-TGAGCATTTTTATATCCATCTCCACCAT 1167 55890- TTGC 56621_438_465_R56621_366_395_F 2082 ERMA_NC002952- TGATCGTTGAGAAGGGATTTGCGAAA 470ERMA_NC002952-55890- TCTTGGCTTAGGATGAAAATATAGTGGT 1143 55890- AGA56621_473_504_R GGTA 56621_374_402_F 2083 ERMA_NC002952-TGCAAAATCTGCAACGAGCTTTGG 480 ERMA_NC002952-55890-TCAATACAGAGTCTACACTTGGCTTAGG 964 55890- 56621_491_520_R AT56621_404_427_F 2084 ERMA_NC002952- TCATCCTAAGCCAAGTGTAGACTCTG 297ERMA_NC002952-55890- TGGACGATATTCACGGTTTACCCACTTA 1266 55890- TA56621_986_615_R TA 56621_489_516_F 2085 ERMA_NC002952-TATAAGTGGGTAAACCGTGAATATCG 231 ERMA_NC002952-55890-TTGACATTTGCATGCTTCAAAGCCTG 1397 55890- TGT 56621_640_665_R56621_586_614_F 2086 ERMC_NC0059_08-2004- TCTGAACATGATAATATCTTTGAAAT 399ERMC_NC005908-2004- TCCGTAGTTTTGCATAATTTATGGTCTA 1041 2738_85_116_FCGGCTC 2738_173_206_R TTTCAA 2087 ERMC_NC005908-2004-TCATGATAATATCTTTGAAATCGGCT 298 ERMC_NC005908-2004-TTTATGGTCTATTTCAATGGCAGTTACG 1429 2738_90_120_F CAGGA 2738_160_189_R AA2088 ERMC_NC005908-2004- TCAGGAAAAGGGCATTTTACCCTTG 283ERMC_NC005908-2004- TATGGTCTATTTCAATGGCAGTTACGA 936 2738_115_139_F2738_61_187_R 2089 ERMC_NC005908-2004- TAATCGTGGAATACGGGTTTGCTA 165ERMC_NC005908-2004- TCAACTTCTGCCATTAAAAGTAATGCCA 956 2738_374_397_F2738_425_452_R 2090 ERMC_NC005908-2004- TCTTTGAAATCGGCTCAGGAAAAGG 421ERMC_NC005908-2004- TGATGGTCTATTTCAATGGCAGTTACGA 1185 2738_101_125_F2738_159_188_R 2091 ERMB_Y13600-625- TGTTGGGAGTATTCCTTACCATTTAA 644ERME_Y13_600-625- TCAACAATCAGATAGATGTCAGACGCAT 953 1362_291_321_F GCACA1362_352_980_R G 2092 ERMB_Y13600-625- TGGAAAGCCATGCGTCTGACATCT 536ERMB_Y13600-625- TGCAAGAGCAACCCTAGTGTTCG 1196 1362_344_67_F1362_415_437_R 2093 ERMB_Y13600-625- TGGATATTCACCGAACACTAGGGTTG 556ERMB_Y13600-625- TAGGATGAAAGCATTCCGCTGGC 919 1362_404_429_F1362_471_393_R 2094 ERMB_Y13600-625- TAAGCTGCCAGCGGAATGCTTTC 161ERMB_Y13600-625- TCATCTGTGGTATGCCGGGTAAGTT 989 1362_465_487_F1362_521_545_R 2095 PVLUK_NC003923- TGAGCTGCATCAACTGTATTGGATAG 456PVLUK_NC003923-1529595- TGGAAAACTCATGAAATTAAAGTGAAAG 1261 1529595-1531285_775_804_R GA 1531285_688_713_F 2096 PVLUK_NC003923-TGGAACAAAATAGTCTCTCGGATTTT 539 PVLUK_NC003923-1529595-TCATTAGGTAAAATGTCTGGACATGATC 993 1529595- GACT 1531285_1095_1125_R CAA1531285_1039_1068_F 2097 PVLUK_NC003923- TGAGTAACATCCATATTTCTGCCATA 461PVLUK_NC003923-1529595- TCTCATGAAAAAGGCTCAGGAGATACAA 1124 1529595- CGT1531285_950_978_R G 1531285_908_936_F 2098 PVLUK_NC003923-TCGGAATCTGATGTTGCAGTTGTT 373 PVLUK_NC003923-1529595-TCACACCTGTAAGTGAGAAAAAGGTTGA 968 1529595- 1531285_654_682_R T1531285_610_633_F 2099 SA442_NC003923- TGTCGGTACACGATATTCTTCACGA 635SA442_NC003923-2538576- TTTCCGATGCAACGTAATGAGATTTCA 1433 2538576-2538831_98_124_R 2538831_11_35_F 2100 SA442_NC003923-TGAAATCTCATTACGTTCCATCGGAA 427 SA4423_NC003923-2538576-TCGTATGACCAGCTTCGGTACTACTA 1098 2538576- A 2538831_163_188_R2538831_98_124_F 2101 SA442_NC003923- TCTCATTACGTTGCATCGGAAACA 395SA442_NC003923-2538576- TTTATGACCAGCTTCGGTACTACTAAA 1428 2538576-2538831_161_187_R 2538831_103_126_F 2102 SA442_NC003923-TAGTACCGAAGCTGGTCATACGA 226 SA442_NC003923-2538576-TGATAATGAAGGGAAACCTTTTTCACG 1179 2538376- 2538831_231_257_R2538831_166_188_F 2103 SEA_NC003923_ TGCAGGGAACAGCTTTAGGCA 495SEA_NC003923-2052219- TCGATCGTGACTCTCTTTATTTTCAGTT 1070 2052219-2051456_173_200_R 2051456_115_135_F 2104 SEA_NC003923-TAACTCTCATGTTTTTCATGGGAAGG 156 SEA_NC003923-2052219-TCTAATTAACCGAAGCTTCTCTACAAGT 1315 2052219- T 2051456_621_651_R ATG2051456_572_598_F 2105 SEA_NC003923- TGTATGGTGGTGTAACGTTACATGAT 629SEA_NC003923-2052219- TAACCGTTTCCAAAGGTACTGTATTTTG 861 2052219- AATAATC2051456_464_492_R T 2051456_382_414_F 2106 SEA_NC003923-TTGTATCTATGGTCCTGTAACCTTAC 695 SEA_NC003923-2052219-TAACCCTTTCCAAAGGTACTCTATTTTG 862 2052219- ATGA 2051456_459_492_R TTTACC2051456_377_406_F 2107 SEB_NC002758- TTTCACATGTAATTTTGATATTCGCA 702SEB_NC002758-2135540- TCATCTGGTTTACGATCTCCTTGACT 988 2135540- CTGA2135140_273_298_R 2135140_208_237_F 2108 SEB_NC002758-TATTTCACATCTAATTTTCATATTCC 244 SEB_NC002758-2135540-TGCAACTCATCTGGTTTAGGATCT 1194 2135540- CACT 2135140_281_304_R2135140_206_235_F 2109 SEB_NC002758- TAACAACTCCCCTTATGAAACGGGAT 151 SEENC002758-2135540- TGTGCAGGCATCATGTCATACCAA 1334 2135540- ATA2135140_402_402_R 2135140_402_402_F 2110 SEB_NC002758-TTGTATGTATGGTGGTGTAACTGACC 696 SEB_NC002758-2135540-TTACCATCTTCAAATACCCGAACACTAA 1361 2135540- A 2135140_402_402_2_R2135140_402_402_2_F 2111 SEC_NC003923- TTAACATGAACGAAACCACTTTGATA 648SEC_NC003923-851678- TCAGTTTGCACTTCAAAAGAAATTGTGT 1177 851678- ATGG852768_620_647_R 852768546_575_F 2112 SEC_NC003923-TGCAATAACAAAACATGAACCAAACC 546 SEC_NC003923-851678-TCAGTTTOCACTTCAAAAGAAATTGTGT 985 851678- ACTT 852768_619_647_R T852768537_566_F 2113 SEC_NC003923- TGACTTTAACAGTTCACCATATGAAA 466SEC_NC003923-851678- TCGCCTGGTCCAGGCATCATAT 1078 851678- CAGG852768_794_815_R 852768_720_749_F 2114 SEC_NC003923-TCGTATGATATGATGCCTGCACCA 604 SEC_NC003923-851678-TCTTCACACTTTTAGAATCAACCGTTTT 1133 851678- 852768_853_886_R ATTGTC852768_787_810_F 2115 SED_M28521_657_682_ TGGTGGTGAAATAGATAGGACTCCTT 615SED_M28521_741_770_R TCTACACCATTTATCCACAAATTGATTG 1318 F GT 2116SED_M28521_690_711_ TCCACCTCTCACTCCACACCAA 554 SED_M28521_739_770_RTCCCCACCATTTATCCACAAATTCATTC 1288 F CTAT 2117 SED_M28521_833_854_TTCCACAACCAACCCCCTATTT 683 SED_M28521_888_911_R TCCCCCTCTATTTTTCCTCCCACA1079 F 2118 SED_M28521_962_987_ TCCATCTTAACCCTCATTTTCCCCAA 559SED_M28521_1022_1048_R TCTCAATATCAACCTCCTCTCTCCATA 1320 F 2119SEA-SEE_NC002952- TTTACACTACTTTTATTCATTCCCCT 699SEA-SEE_NC002952-2131289- TCATTTATTTCTTCCCTTTTCTCCCTAC 994 2131289- AACC2130703_71_98_R 2130703_16_45_F 2120 SEA-SEE_NC002_952-TCATCATCCCTCCTATAACCATTTAT 469 SEA-SEE_NC002952-2131289-TAACCACCATATAACTCTACTTTTTTCC 870 2131289- TACT 2130703_314_344_R CTT2130703_249_278_F 2121 SEE_NC002952- TGACATGATAATAACCGATTGACCGA 445SEE_NC002952-2131289- TCTATAGGTACTGTAGTTTGTTTTCCGT 1120 2131289- AGA2130703_465_494_R CT 2130703_409_437_F 2122 SEE_NC002952-TGTTCAAGAGCTAGATCTTCAGGCAA 640 SEE_NC002952-2131289-TTTGCACCTTACCGCCAAAGCT 1436 2131289- 2130703_586_586_R 2130703_525_550_F2123 SEE_N4C002952- TGTTCAAGAGCTAGATCTTCAGGCA 639 SEE_NC002952-2131289-TACCTTACCGCCAAAGCTGTCT 892 2131289- 2130703_586_586_2_R2130703_525_549_F 2124 SEE_NC002952- TCTGGAGGCACACCAAATAAAACA 403SEE_NC002952-2131289- TCCGTCTATCCACAAGTTAATTGGTACT 1043 2131289-2130703_444_471_R 2130703_361_384_F 2125 SEG_NC002758-TGCTCAACCCGATCCTAAATTAGACG 520 SEG_NC002758-1955100-TAACTCCTCTTCCTTCAACAGGTGGA 863 1955100- A 1954171_321_346_R1954171_225_251_F 2126 SEG_NC002758- TGGACAATAGACAATCACTTGGATT 548SEG_NC002758-1955100- TGCTTTGTAATCTAGTTCCTGAATAGTA 1260 1955100- ACA1954171_671_702_R ACCA 1954171_623_651_F 2127 SEG_NC002758-TGGAGGTTGTTGTATGTATGGTGGT 555 SEG_NC0002758-1955100-TGTCTATTGTCGATTGTTACCTGTACAG 1329 1955100- 1954171_607_635_R T1954171_540_564_F 2128 SEG_NC002758- TACAAAGCAAGACACTGGCTCACTA 173SEG_NC002758-1955100- TGATTCAAATGCAGAACCATCAAACTCG 1187 1955100-1954171_735_762_R 1954171_694_718_F 2129 SEH_NC002953-60024-TTGCAACTGCTGATTTAGCTCAGA 682 SEH_NC002953-60024-TAGTGTTGTACCTCCATATAGACATTCA 927 60977_449_472_F 60977_547_576_R GA 2130SEH_NC002953-60024- TAGAAATCAAGGTGATAGTGGCAATG 201 SEH_EC002953-60024-TTCTGAGCTAAATCAGCAGTTGCA 1390 60977_408_434_F A 60977_450_473_R 2131SEH_NC0029S3-60024- TCTGAATGTCTATATGGAGGTACAAC 400 SEH_NC002953-60024-TACCATCTACCCAAACATTAGCACCAA 888 60977_547_576_F ACTA 60977_608_634_R2132 SEH_NC002953-60024- TTCTGAATGTCTATATGGAGGTACAA 677SEH_NC002953-60024- TAGCACCAATCACCCTTTCCTGT 909 60977_546_575_F CACT60977_594_616_R 2133 SEH_NC002758- TCAACTCGAATTTTCAACAGGTACCA 253SEH_NC002758-1957830- TCACAAGGACCATTATAATCAATGCCAA 966 1957830-1956949_419_446_R 1956949_324_349_F 2134 SEI_NC002758-TTCAACAGGTACCAATGATTTGATCT 666 SEI_NC002758-1957830-TGTACAAGGACCATTATAATCAATGCCA 1316 1957830- CA 1956949_420_447_R1956949_336_363_F 2135 SEI_NC002758- TGATCTCAGAATCTAATAATTGGGAC 471SEI_NC002758-1957830- TCTGGCCCCTCCATACATGTATTTAG 1129 1957830- GAA1956949_449_474_R 1956949_356_384_F 2136 SEI_NC002758-TCTCAAGGTGATATTGGTGTAGGTAA 394 SEI_NC002758-1957830-TGGGTAGGTTTTTATCTGTGACGCCTT 1293 1957830- CTTAA 1956949_290_316_R1956949_223_253_F 2137 SEJ_AF053140_1307_1 TGTGGAGTAACACTGCATGAAAACAA637 SEJ_AF053140_1381_1404_R TCTAGCGGAACAACAGTTCTGATG 1118 332_F 2138SEJ_AF053140_1378_1 TAGCATCAGAACTGTTGTTCCGCTAG 211SEJ_AF053140_1429_1458_R TCCTGAAGATCTAGTTCTTGAATGGTTA 1049 403_F CT 2139SEJ_AF053140_1431_1 TAACCATTCAAGAACTAGATCTTCAG 153SEJ_AF053140_1500_1531_R TAGTCCTTTCTGAATTTTACCATCAAAG 925 459_F GCA GTAC2140 SEJ_AF053140_1434_1 TCATTCAAGAACTAGATCTTCAGGCA 301SEJ_AF053140_1521_1549_R TCAGGTATGAAACACGATTAGTCCTTTC 984 461_F AG T2141 TSST_NC002758- TGGTTTAGATAATTCCTTAGGATCTA 619TSST_NC002758-2137564- TGTAAAAGCAGGGCTATAATAAGGACTC 1312 2137564- TGCGT2138293_278_305_R 2138293_206_236_F 2142 TSST_NC002758-TGCGTATAAAAAACACAGATGGCAGC 514 TSST_NC002758-2137564-TGCCCTTTTGTAAAAGCAGGGCTAT 1221 2137564- A 2138293_289_313_R2138293_232_258_F 2143 TSST_NC002758- TCCAAATAAGTGGCGTTACAAATACT 304TSST_NC002758-2137564- TACTTTAAGGGGCTATCTTTACCATGAA 907 2137564- GAA2138293_448_478_R CCT 2138293_382_410_F 2144 TSST_NC002758-TCTTTTACAAAAGGGGAAAAAGTTGA 423 TSST_NC002758-2137564-TAAGTTCCTTCGCTAGTATGTTGGCTT 874 2137564- CTT 2138293_347_373_R2138293_297_325_F 2145 ARCC_NC003923- TCGCCGGCAATGCCATTGGATA 368ARCC_NC003923- TGAGTTAAAATGCGATTGATTTCAGTTT 1175 2725050-2724595_97_128_R CCAA 2724595_37_58_F 2146 ARCC_NC003923-TGAATAGTGATAGAACTGTAGGCACA 437 ARCC_NC003923-2725050-TCTTCTTCTTTCGTATAAAAAGGACCAA 1137 2725050- ATCGT 2724595_214_245_R TTGG2724595_131_161_F 2147 ARCC_NC003923- TTGGTCCTTTTTATACGAAAGAAGAA 691ARCC_NC003923-2725050- TGGTGTTCTAGTATAGATTGAGGTAGTG 1306 2725050- GTTGAA2724595_322_353_R GTGA 2724595_218_249_F 2148 AROE_NC003923-TTGCGAATAGAACGATGGCTCGT 686 AROE_NC003923-1674726-TCGAATTCAGCTAAATACTTTTCAGCAT 1064 1674726- 1674277_435_464_R CT1674277_371_393_F 2149 AROE_NC003923- TGGGGCTTTAAATATTCCAATTGAAG 590AROE_NC003923-1674726- TACCTGCATTAATCGCTTGTTCATCAA 891 1674726- ATTTTCA1674277_155_181_R 1674277_30_62_F 2150 AROE_NC003923-TGATGGCAAGTGGATAGGGTATAATA 474 ARCE_NC003923-1674726-TAAGCAATACCTTTACTTGCACCACCTG 869 1674726- CAG 1674277_308_335_R1674277_204_232_F 2151 GLPF_NC003923- TGCACCGGCTATTAAGAATTACTTTG 491GLPF_NC003923-1296927- TGCAACAATTAATGCTCCGACAATTAAA 1193 1296927- CCAACT1297391_382_414_R GGATT 1297391_270_301_F 2152 GLPF_NC003923-TGGATGGGGATTAGCGGTTACAATG 558 GLPF_NC003923-1296927-TAAAGACACCGCTGGGTTTAAATGTGCA 850 1296927- 1297391_81 _108_R1297391_27_51_F 2153 GLPF_NC003923- TAGCTGGCGCGAAATTAGGTGT 218GLPF_NC003923-1296927- TCACCGATAAATAAAATACCTAAAGTTA 972 1296927-1297391_323_359_R ATGCCATTG 1297391_239_260_F 2154 GMK_NC003923-TACTTTTTTAAAACTAOGGATGCGTT 200 GMK_NC003923-1190906-TGATATTGAACTGGTGTACCATAATAGT 1180 1190906- TGAAGC 1191334_166_197_R TGCC1191334_91_122_F 2155 GMK_NC003923- TGAAGTAGAAGGTGCAAAGCAAGTTA 435GMK_14C003923-1190906- TCGCTCTCTCAAGTGATCTAAACTTGGA 1082 1190906- GA1191334_305_333_R G 1191334_240_267_F 2156 GMK_NC003923-TCACCTCCAAGTTTAGATCACTTGAG 288 GMK_NC003923-1190908-TGGGACGTAATCGTATAAATTCATCATT 1284 1190906- AGA 1191334_403_432_R TC1191334_301_329_F 2157 PTA_NC003923- TCTTGTTTATGCTGGTAAAGCAGATG 418PTA_NC003923-628885- TGGTACACCTGGTTTCGTTTTGATGATT 1301 628885- G629355_314_345_R TGTA 629355_237_263_F 2158 PTA_NC003923-TGAATTAGTTCAATCATTTGTTGAAC 439 PTA_NC003923-628885-TGCATTGTACCGAAGTAGTTCACATTGT 1207 628885- GACGT 629355_211_239_R T629355_141_171_F 2159 PTA_NC003923- TCCAAACCAGGTGTATCAAGAACATC 303PTA_NC003923-628885- TGTTCTGGATTGATTGCACAATCACCAA 1349 628885- AGG629355_393_422_R AG 629355_328_356_F 2160 TPI_NC003923-TGCAAGTTAAGAAAGCTGTTGCAGGT 486 TPI_NC003923-830671-TGAGATGTTGATGATTTACCAGTTCCGA 1165 830671- TTAT 831072_209_239_R TTG831072_131_160_F 2161 TPI_NC003923- TCCCACGAAACAGATGAAGAAATTAA 318TPI_NC003923-830671- TGGTACAACATCGTTAGCTTTACCACTT 1300 830671- CAAAAAAG831072_97_129_R TCACG 831072_1_34_F 2162 TPI_NC003923-TCAAACTGGGCAATCGGAACTGGTAA 246 TPI_NC003923-830671-TGGCAGCAATAGTTTGACGTACAAATGC 1275 830671- ATC 831072_253_286_R ACACAT831072_199_227_F 2163 YQI_NC003923- TGAATTGCTGCTATGAAAGGTGGCTT 440YQI_NC003923-378916- TCGCCAGCTAGCACGATGTCATTTTC 1076 378916-379431_259_284_R 379431_142_167_F 2164 YQI_NC003923-TACAACATATTATTAAAGAGACGGGT 175 YQI_NC003923-378916-TTCGTGCTGGATTTTGTCCTTGTCCT 1388 378916- TTGAATCC 379431_120_145_R379431_44_77_F 2165 YQI_NC003923- TCCAGCACGAATTGCTGCTATGAAAG 314YQI_NC003923-378916- TCCAACCCAGAACCACATACTTTATTCA 997 378916-379431_193_221_R C 379431_135_160_F 2166 YQI_NC003923-TAGCTGGCGGTATGGAGAATATGTCT 219 YQI_NC003923-378916-TCCATCTGTTAAACCATCATATACCATG 1013 378916- 379431_364_396_R CTATC379431_275_300_F 2167 BLAZ_(1913827 . . . 19 TCCACTTATCGCAAATGGAAAATTAA312 BLAZ_(1913827 . . . 1914672)_(—) TGGCCACTTTTATCAGCAACCTTACAGT 127714672)_546_575_F GCAA 655_683_R C 2168 BLAZ_(1913827 . . . 19TGCACTTATCGCAAATGGAAAATTAA 494 BLAZ_(1913827 . . . 1914672)_(—)TAGTCTTTTGGAACACCGTCTTTAATTA 926 14672)_546_575_2_F GCAA 628_659_R AAGT2169 BLAZ_(1913827 . . . 19 TGATACTTCAACGCCTGCTGCTTTC 467 BLAZ_(1913827. . . 1914672)_(—) TGGAACACCGTCTTTAATTAAAGTATCT 1263 14672)_507_531_F622_651_R CC 2170 BLAZ_(1913827 . . . 19 TATACTTCAACGCCTGCTGCTTTC 232BLAZ_(1913827 . . . 1914672)_(—) TCTTTTCTTTGCTTAATTTTCCATTTGC 114514672)_508_531_F 553_583_R GAT 2171 BLAZ_(1913827 . . . 19TGCAATTGCTTTAGTTTTAAGTGCAT 487 BLAZ_(1913827 . . . 1914672)_(—)TTACTTCCTTACCACTTTTAGTATCTAA 1366 14672)_24_56_F GTAATTC 121_154_RAGCATA 2172 BLAZ_(1913827 . . . 19 TCCTTGCTTTAGTTTTAAGTGCATGT 351BLAZ_(1913827 . . . 1914672)_(—) TGGGGACTTCCTTACCACTTTTAGTATC 128914672)_26_58_F AATTCAA 127_157_R TAA 2173 BLAZ_NC002952-TCCACTTATCGCAAATGGAAAATTAA 312 BLAZ_NC002952-1913827-TGGCCACTTTTATCAGCAACCTTACAGT 1277 1913827- GCAA 1914672_655_683_R C1914672_546_575_F 2174 BLAZ_NC002952- TGCACTTATCGCAAATGGAAAATTAA 494BLAZ_NC002952-1913827- TAGTCTTTTGGAACACCGTCTTTAATTA 926 1913827- GCAA1914672_628_659_R AAGT 1914672_546_575_2_F 2175 BLAZ_NC002952-TGATACTTCAACGCCTGCTGCTTTC 467 BLAZ_NC002952-1913827-TGGAACACCGTCTTTAATTAAAGTATCT 1263 1913827- 1914672_622_651_R CC1914672_507_531_F 2176 BLAZ_NC002952- TATACTTCAACGCCTGCTGCTTTC 232BLAZ_NC002952-1913827- TCTTTTCTTTGCTTAATTTTCCATTTGC 1145 1913827-1914672_553_583_R GAT 1914672_08_531_F 2177 BLAZ_NC002952-TGCAATTGCTTTAGTTTTAAGTGCAT 487 BLAZ_NC002952-1913827-TTACTTCCTTACCACTTTTAGTATCTAA 1366 1913827- GTAATTC 1914672_121_154_RAGCATA 1914672_24_56_F 2178 BLAZ_NC002952- TCCTTGCTTTAOTTTTAAGTGCATGT351 BLAZ_NC002952-1913827- TGGGGACTTCCTTACCACTTTTACTATC 1289 1913827-AATTCAA 1914672_127_157_R TAA 1914672_26_58_F 2247 TUFB_NC002758-TGTTGAACGTGGTCAAATCAAACTTC 643 TUFBNC002758-615038-TGTCACCAGCTTCAGCGTAGTCTAATAA 1321 615038- GTG 616222_793_820_R616222_693_721_F 2248 TUFB_NC002758- TCGTGTTGAACGTGGTCAAATCAAAG 386TUFB_NC002758-615038- TGTCACCAGCTTCAGCGTAGTCTAATAA 1321 615038- T616222_793_820_R 616222_690_716_F 2249 TUFB_NC002758-TGAACGTGGTCAAATCAAAGTTGGTG 430 TUFB_NC002758-615038-TGTCACCAGCTTCAGCGTAGTCTAATAA 1321 615038- AAGA 616222_793_820_R616222_696_725_F 2250 TUFB_NC002758- TCCCAGGTGACGATGTACCTGTAATC 320TUFB_NC-02758-615038- TGGTTTGTCAGAATCACGTTCTGGAGTT 1311 615038-616222_601_630_R GG 616222_488_513_F 2251 TUFB_NC002758-TGAAGGTGGACGTCACACTCCATTCT 433 TUFB_NC002758-615038-TAGGCATAACCATTTCAGTACCTTCTGG 922 615038- TC 616222_1030_1060_R TAA616222_945_972_F 2252 TUFB_NC002758- TCCAATGCCACAAACTCGTGAACA 307TUFB_NC002758-615038- TTCCATTTCAACTAATTCTAATAATTCT 1382 615038-616222_424_459_R TCATCGTC 616222_333_356_F 2253 NUC_NC002758-TCCTGAAGCAAGTGCATTTACGA 342 NUC_NC002758-894288-TACGCTAAGCCACGTCCATATTTATCA 899 894288- 894974_483_509_R894974_402_424_F 2254 NUC_NC002758- TCCTTATAGGGATGGCTATCAGTAAT 349NUC_NC002758-894288- TGTTTGTGATGCATTTGCTGAGCTA 1354 894288- GTT894974_165_189_R 894974_53_81_F 2255 NUC_NC002758-TCAGCAAATGCATCACAAACAGATAA 273 NUC_NC002758-894288-TAGTTGAAGTTGCACTATATACTGTTGG 928 894288- 894974_222_250_R A894974_169_194_F 2256 NUC_NC002758- TACAAACCTCAACCAATCACATTCAC 174NUC_NC002758-894288- TAAATCCACTTCCTTCACCCCCATAT 853 894288- ACTA894974_396_421_R 894974_316_345_F 2270 RPOB_EC_3798_3821_1_TCCCCACCCCTTCCCTCAAATCCA 566 RPOB_EC_3868_3895_RTCACCTCCTCCCACTTCACCCTCACCAT 979 F 2271 RPOB_EC_3789_3812_FTCACTTCCCCCCTCACCCCTTCCC 294 RPOB_EC_3860_3890_RTCGTCCCACTTAACCCTCACCATTTCCT 1107 CCA 2272 RPOB_EC_3789_3812_FTCACTTCCCCCCTCACCCCTTCCC 294 RPOB_EC_3860_3890_2_RTCCTCCCACTTAACCCTCACCATTTCCT 1102 CCA 2273 RPOB_EC_3789_3812_FTCACTTCCCCCCTCACCCCTTCCC 294 RPOB_EC_3862_3890_RTCCTCCCACTTAACCCTCACCATTTCCT 1106 G 2274 RPOB_EC_3789_3812_FTCACTTCCCCCCTCACCCCTTCGC 294 RPOB_EC_3862_3890_2_RTCCTCCCACTTAACCCTCACCATTTCCT 1101 C 2275 RPOB_EC_3793_3812_FTTCCCCCCTCACCCCTTCCC 674 RPOB_EC_3865_3890_R TCCTCCCACTTAACCCTCACCATTTC1105 2276 RPCB_EC_3793_3812_F TTCCCCCCTCACCCCTTCCC 674RPOB_EC_3865_3890_2_R TCCTCCCACTTAACCCTCACCATTTC 1100 2309MUPR_X75439_1658_16 TCCTTTCATATATTATCCCATCCAAC 352MUFR_X75439_1744_1773_R TCCCTTCCTTAATATCACAACCAAACCA 1030 89_F CTTCCT CT2310 MUPR_X75439_1330_13 TTCCTCCTTTTCAAACCCACCCTT 669MUPR_X75439_1413_1441_R TCACCTCCTCCTATATCAACAATACCAC 1171 53_F T 2312MUPR_X75439_1314_13 TTTCCTCCTTTTCAAACCCACCCTT 704MUPR_X75439_1381_1409_R TATATCAACAATACCACTTCCTTCTCAC 931 38_F T 2313MUPR_X75439_2486_25 TAATTCCCCTCTTTCTCCCTTAAACA 172MUPR_X75439_2548_2574_R TTAATCTCCCTCCCCAACTCAAATCCT 1360 16_F CCTTA 2314MUPR_X75439_2547_25 TACCATTTCACTTCCCCACCCACATT 188MUPR_X75439_2605_2630_R TCCTCCTCTCCAATCTCCCATATACC 1103 72_F 2315MUPR_X75439_2666_26 TCCCTACAATACCCTTTATCAAATTT 513MUPR_X75439_2711_2740_R TCACATATAAATCCAACAAATCCACCCA 981 96_F TAACA CT2316 MUPR_X75439_2813_28 TAATCAACCATTCCAACATCAAATCC 165MUPR_X75439_2867_2890_R TCTCCATTTTTCCCACCCTCTCTA 1127 43_F ATACC 2317MUPR_X75439_884_914_ TCACATCCACTCCCCCTATATAACTC 447MUPR_X75439_977_1007_R TCTACAATAACCACTCACCTTATCTCCC 1317 F TTCAC TTA2318 CTXA_NC002505- TCCTCTTATCCCAACACCACACACTC 608CTXA_NC002505-1568114- TCCTCCCTAACAAATCCCCTCTCACTTC 1109 1568114- ACT1567341_194_221_R 1567341_114_142_F 2319 CTXA_NC002505-TCTTATCCCAACACCACACACTCACT 411 CTXA_NC002505-1568114-TCCTCCCTAACAAATCCCCTCTCACTTC 1109 1568114- ACT 1567341_194_221_R1567341_117_145_F 2320 CTXA_NC002505- TCCTCTTATCCCAACACCACACACTC 608CTXA_NC002505-1568114- TAACAAATCCCCTCTCACTTCCTCTTCC 855 1568114- ACT1567341_186_214_R A 1567341_114_142_F 2321 CTXA_NC002505-TCTTATCCCAACACCACACACTCACT 411 CTXA_NC002505-1568114-TAACAAATCCCCTCTCACTTCCTCTTCC 855 1568114- ACT 1567341_186_214_R A1567341_117_145_F 2322 CTXA_NC002505- ACCACACACTCACTACTTTCACCCAC 27CTXA_NC002505-1568114- TCCCCTCTCACTTCCTCTTCCATCATCA 1027 1568114- CT1567341_180_207_R 1567341_129_156_F 2323 CTXA_NC002505-TGCCAAGAGGACAGAGTGAGTACTTT 500 CTXA_NC002505-1568114-TAACAAATCCCGTCTGAGTTCCTCTTGC 855 1568114- GA 1567341_186_214_R A1567341_122_49_F 2324 INV_U22457-74- TGCTTATTTACCTGCACTCCCACAAC 530INV_U22457-74- TGACCCAAAGCTGAAAGCTTTACTG 1154 3772_831_858_F TG3772_942_966_R 2325 INV_U22457-74- TGAATGCTTATTTACCTGCACTCCCA 438INV_U22457-74- TAACTGACCCAAAGCTGAAAGCTTTACT 864 3772_827_857_F CAACT3772_942_970_R G 2326 INV_U22457-74- TGCTGGTAACAGAGCCTTATAGGCGC 526INV_U22457-74- TGGGTTGCGTTGCAGATTATCTTTACCA 1296 3772_1555_1581_F A3772_619_1647_R A 2327 INV_U22457-74- TGGTAACAGAGCCTTATAGGCGCATA 598INV_U22457-74- TCATAAGGGTTGCGTTGCAGATTATCTT 987 3772_1558_1585_F TG3772_1622_1652_R TAC 2328 ASD_NC006570- TGAGGGTTTTATGCTTAAAGTTGGTT 459ASD_NC006570-439714- TGATTCGATCATACGAGACATTAAAACT 1188 439714- TTATTGGTT438608_54_84_R GAG 438608_937_F 2329 ASD_NC006570-TAAAGTTGGTTTTATTGGTTGGCGCG 149 ASD_EC006570-439714-TCAAAATCTTTTGATTCGATCATACGAG 948 439714- GA 438608_66_95_R AC438608_18_45_F 2330 ASD_NC006570- TTAAAGTTGGTTTTATTGGTTGGCGC 647ASD_NC006570-439714- TCCCAATCTTTTGATTCGATCATACGAG 1016 439714- GGA438608_67_95_R A 438608_17_45_F 2331 ASD_NC006570-TTTTATGCTTAAAGTTGGTTTTATTG 709 ASD_NC006570-439714-TCTGCCTGAGATGTCGAAAAAAACGTTG 1128 439714- GTTGGG 438608_07_134_R438608_9_40_F 2332 GALE_AF513299_171_2 TCAGCTAGACCTTTTAGGTAAAGCTA 280GALE_AF513299_241_271_R TCTCACCTACAGCTTTAAAGCCAGCAAA 1122 00_F AGCT ATG2333 GALE_AF513299_168_1 TTATCAGCTAGACCTTTTAGGTAAAG 658GALE_AF513299_245_271_R TCTCACCTACAGCTTTAAAGCCAGCAA 1121 99_F CTAAGC2334 GALE_AF513299_168_1 TTATCAGCTAGACCTTTTAGGTAAAG 658GALE_AF513299_233_264_R TACAGCTTTAAAGCCAGCAAAATGAATT 883 99_F CTAAGCACAG 2335 GALE_AF513299_169_1 TCCCAGCTAGACCTTTTAGGTAAAGC 319GALE_AF513299_252_279_R TTCAACACTCTCACCTACAGCTTTAAAG 1374 98_F TAAG 2336PLA_AF053945_7371_7 TTGAGAAGACATCCGGCTCACGTTAT 680PLA_AF053945_7434_7468_R TACGTATGTAAATTCCGCAAAGACTTTG 900 403_F TATGGTAGCATTAG 2337 PLA_AF053945_7377_7 TGACATCCGGCTCACGTTATTATGGT 443PLA_AF053945_7428_7455_R TCCGCAAAGACTTTGGCATTAGGTGTGA 1035 403_F A 2338PLA_AF053945_7377_7 TGACATCCGGCTCACGTTATTATGGT 444PLA_AF05394S_7430_7460_R TAAATTCCGCAAAGACTTTGGCATTAGG 854 404_F AC TGT2339 CAF_AF053947_33412_ TCCGTTATCGCCATTGCATTATTTGG 329CAF_AF053947_33498_33523_ TAAGAGTGATGCGGGCTGGTTCAACA 866 33441_F AACT R2340 CAF_AF053947_33426_ TGCATTATTTGGAACTATTGCAACTG 499CAF_AF053947_33483_33507_ TGGTTCAACAAGAGTTGCCGTTGCA 1308 33458_F CTAATGCR 2341 CAF_AF053947_33407 TCAGTTCCGTTATCGCCATTGCA 291CAF_AF053947_33483_33504_ TTCAACAAGAGTTGCCGTTGCA 1373 33429_F R 2342CAF_AF053947_33407 TCAGTTCCGTTATCGCCATTGCATT 293CAF_AF053947_33494_33517_ TGATGCGGGCTGGTTCAACAAGAG 1184 33431_F R 2344GAPA_NC_002505_1_28_ TCAATGAACGATCAACAAGTGATTGA 260GAPA_NC_002505_29_58_R_1 TCCTTTATGCAACTTGGTATCALCAGGA 1060 F_1 TG AT2472 OMPA_NC000117_68_89_ TGCCTGTAGGGAATCCTGCTGA 507OMPA_NC000117_145_167_R TCACACCAAGTAGTGCAAGGATC 967 F 2473OMPA_NC000117_798_8 TGATTACCATGAGTGGCAAGCAAG 475 OMPA_NC000117_865_893_RTCAAAACTTGCTCTAGACCATTTAACTC 947 21_F C 2474 OMPA_NC000117_645_6TGCTCAATCTAAACCTAAAGTCGAAG 521 OMPA_NC000117_757_777_RTGTCGCAGCATCTGTTCCTGC 1328 71_F A 2475 OMPA_NC000117_947_9TAACTGCATGGAACCCTTCTTTACTA 157 OMPA_NC000117_1011_1040_RTGACAGGACACAATCTGCATGAAGTCTG 1153 73_F C AG 2476 OMPA_NC000117_774_7TACTGGAACAAAGTCTGCGACC 196 OMPA_NC000117_871_894_RTTCAAAAGTTGCTCGAGACCATTG 1371 95_F 2477 OMPA_NC000117_457_4TTCTATCTCGTTGGTTTATTCGGAGT 676 OMPA_NC000117_511_534_RTAAAGAGACGTTTGGTAGTTCATTTGC 851 83_F T 2478 OMPA_NC000117_687_7TAGCCCAGCACAATTTGTGATTCA 212 OMPA_NC000117_787_816_RTTGCCATTCATGGTATTTAAGTGTAGCA 1406 10_F GA 2479 OMPA_NC000117_540_5TGGCGTAGTAGAGCTATTTACAGACA 571 OMPA_NC000117_649_672_RTTCTTGAACGCGAGGTTTCGATTG 1395 66_F C 2480 OMPA_NC000117_338_3TGCACGATGCGGAACGGTTCACA 492 OMPA_NC000117_417_444_RTCCTTTAAAATAACCGCTAGTAGCTCCT 1058 60_F 2481 OMP2_NC000117_1840_TATGACCAAACTCATCAGACGAG 234 OMP2_NC000117_71_91_R TCCCGCTGGCAAATAAACTCG1025 F 2482 OMP2_NC000117_354_3 TGCTACGGTAGGATCTCCTTATCCTA 516OMP2_NC000117_445_471_ TGGATCACTGCTTACGAACTCAGCTTC 1270 82_F TTG 2483OMP2_NC000117_1297 TGGAAAGGTGTTGCAGCTACTCA 537 OMP2_NC000117_1396_1419_RTACGTTTGTATCTTCTCCAGAACC 903 1319_F 2484 OMP2_NC00011714_65TCTGGTCCAACAAAAGGAACGATTAC 407 OMP2_NC000117_1541_1569_RTCCTTTCAATGTTACAGAAAACTCTACA 1062 1493_F AGG G 2485 OMP2_NC000117_44_66TGACGATCTTCGCGGTGACTAGT 450 OMP2_NC000117_120_148_RTGTCAGCTAAGCTAATAACGTTTGTAGA 1323 F G 2486 OMP2_NC000117_166_1TGACAGCGAAGAAGGTTAGACTTGTC 441 OMP2_NC000117_240_261_RTTGACATCGTCCCTCTTCACAG 1396 90_F C 2487 GYRA_NC000117_514_5TCAGGCATTGCGCTTGGGATGGC 287 GYRA_NC000117_640_660_RTGCTGTAGGGAAATCAGGGCC 1251 36_F 2488 GYRA_NC000117_801_8TGTGAATAAATCACGATTGATTGAGC 636 GYRA_NC000117_871_893_RTTGTCAGACTCATCGCGAACATC 1419 27_F A 2489 GYRA_NC002952_219_2TGTCATGGGTAAATATCACCCTCA 632 GYRA_NC002952_319_345_RTCCATCCATAGAACCAAAGTTACCTTG 1010 42_F 2490 GYRA_NC002952_964_9TACAAGCACTCCCAGCTGCA 176 GYRA_NC002952_1024_1041_R TCGCAGCGTGCGTGGCAC1073 83_F 2491 GYRA_NC002952_1505_ TCGCCCGCGAGGACGT 366GYRA_NC002952_1546_1562_R TTGGTGCGCTTGGCGTA 1416 1520_F 2492GYRA_NC002952_59_81_ TCAGCTACATCGACTATGCGATG 279 GYRA_NC002952_124_143_RTGGCGATGCACTGGCTTGAG 1279 F 2493 GYRA_NC002952_216_2TGACGTCATCGGTAAGTACCACCC 452 GYRA_NC002952_313_333_RTCCGAAGTTGCCCTGGCCGTC 1032 39_F 2494 GYRA_NC002952_219_2TGTACTCGGTAAGTATCACCCGCA 625 GYRA_NC002952_308_330_RTAAGTTACCTTGCCCGTCAACCA 873 422_F 2495 GYRA_NC002952_115_1TGAGATGGATTTAAACCTGTTCACCG 453 GYRA_NC002952_220_242_RTGCGGGTGATACTTACCGAGTAC 1236 41_F C 2496 GYRA_NC002952_517_5TCAGGCATTGCGGTTGGGATGGC 287 GYRA_NC002952_643_663_RTGCTGTAGGGAAATCAGGGCC 1251 39_F 2497 GYRA_NC002952_273_2TCGTATGGCTCAATGGTGGAG 380 GYRA_NC002952_338_360_RTGCGGCAGCACTATCACCATCCA 1234 93_F 2498 GYRA_NC000912_257_2TGAGTAACTTCCACCCGCACGG 462 GYRA_NC000912_346_370_RTCGAGCCGAAGTTACCCTGTCCGTC 1067 78_F 2504 ARCC_NC003923-TAGTpGATpAGAACpTpGTAGGCpAC 229 ARCC_NC003923-2725050-TCpTpTpTpCpGTATAAAAAGGACpCpA 1116 2725050- pAATpCpCT 2724595_214_239P_RATpTpGG 2724595_135_161P_F 2505 PTA_NC003923- TCTTGTpThTpATGCpTpGGTmGC417 PTA_NC003923-628885- TACpACpCpTGGTpTpTpCpGTpTpTpT 904 628885- AGATGC629355_314_342P_R pGATGATpTpTpGTA 629355_237_263P_F 2517CJMLST_ST1_1852_188 TTTGCGGATGAAGTAGGTGCCTATCT 708CJMLST_ST1_1945_1977_R TGTTTTATGTGTAGTTGAGCTTACTACA 1355 3_F TTTTCCTGAGC 2518 CJMLST_ST1_2963_299 TGAAATTGCTACAGGCCCTTTAGGAC 428CJMLST_ST1_3073_3097_R TCCCCATCTCCGCAAAGACAATAAA 1020 2_F AAGG 2519CJMLST_ST1_2350_237 TGCTTTTGATGGTGATGCAGATCGTT 535CJMLST_ST1_2447_2481_R TCTACAACACTTGATTGTAATTTGCCTT 1117 8_F TGG GTTCTTT2520 CJMLST_ST1_654_684_ TATGTCCAAGAAGCATAGCAAAAAAA 240CJMLST_ST1_725_756_R TCGGAAACAAAGAATTCATTTTCTGGTC 1084 F CCAAT CAAA 2521CJMLST_ST1_360_395_ TCCTGTTATTCCTGAAGTAGTTAATC 347 CJMLST_ST1_454_487_RTGCTATATGCTACAACTGGTTCAAAAAC 1245 F AAGTTTGTTA ATTAAG 2522CJMLST_ST1_1231_125 TGGCAGTTTTACAAGGTGCTGTTTCA 564CJMLST_ST1_1312_1340_R TTTAGCTACTATTCTAGCTGCCATTTCC 1427 8_F TC A 2523CJMLST_ST1_3543_357 TGCTGTAGCTTATCGCGAAATGTCTT 529CJMLST_ST1_3656_3685_R TCAAAGAACCAGCACCTAATTCATCATT 950 4_F TGATTT TA2524 CJMLST_ST1_1_17_F TAAAACTTTTGCCGTAATGATGGGTG 145 CJMLST_ST1_55_84_RTGTTCCAATAGCAGTTCCGCCCAAATTG 1348 AAGATAT AT 2525 CJMLST_ST1_1312_134TGGAAATGGCAGCTAGAATAGTAGCT 538 CJMLST_ST1_1383_1417_RTTTCCCCGATCTAAATTTGGATAAGCCA 1432 2_F AAAAT TAGGAAA 2526CJMLST_ST1_2254_228 TGGGCCTAATGGGCTTAATATCAATG 582CJMLST_ST1_2352_2379_R TCCAAACGATCTGCATCACCATCAAAAG 996 6_F AAAATTG 2527CJMLST_ST1_1380_141 TGCTTTCCTATGGCTTATCCAAATTT 534CJMLST_ST1_1486_1520_R TGCATGAAGCATAAAAACTCTATCAAGT 1205 1_F AGATCGGCTTTTA 2528 CJMLST_ST1_3413_343 TTGTAAATGCCGCTGCTTCAGATCC 692CJMLST_ST1_3511_3542_R TGCTTGCTCAAATCATCATAAACAATTA 1257 7_F AACC 2529CJMLST_ST1_1130_115 TACGCGTCTTGAAGCGTTTCGTTATG 189CJMLST_ST1_1203_1230_R TAGGATGAGCATTATCAGGGAAAGAATC 920 6_F A 2530CJMLST_ST1_2840_287 TGGGGCTTTGCTTTATAGTTTTTTAC 591CJMLST_ST1_2940_2973_R TAGCGATTTCTACTCCTAGAGTTGAAAT 917 2_F ATTTAAGTTCAGG 2531 CJMLST_ST1_2058_208 TATTCAAGGTGGTCCTTTGATGCATG 241CJMLST_ST1_2131_2162_R TTGGTTCTTACTTSTTTTGCATAAACTT 1417 4_F T TCCA 2532CJMLST_ST1_553_585_ TCCTGATGCTCAAAGTGCTTTTTTAG 344 CJMLST_ST1_655_685_RTATTGCTTTTTTTGCTATGCTTCTTGGA 642 F ATCCTTT CAT 2564 GLTA_NC002163-TCATGTTGAGCTTAAACCTATAGAAG 299 GLTA_NC002163-1604930-TTTTGCTCATGATCTGCATGAAGCATAA 1443 1604930- TAAAAGC 1604529_352_380_R A1604529_306_338_F 2565 UNCA_NC002163- TCCCCCACGCTTTAATTGTTTATGAT 322UNCA_NC002163-112166- TCGACCTGGAGGACGACGTAAAATCA 1065 112166- GATTTGAG112647_146_171_R 112647_80_113_F 2566 UNCA_NC002163-TAATGATGAATTAGGTGCGGGTTCTT 170 UNCA_NC002163-112166-TGGGATAACATTGGTTGGAATATAAGCA 1285 112166- T 112647_294_329_R GAAACATC112647_233_259_F 2567 PGM_NC002163- TCTTGATACTTGTAATGTGGGCGATA 414PGM_NC002163-327773- TCCATCGCCAGTTTTTGCATAATCGCTA 1012 327773- AATATGT328270_365_396_R AAAA 328270_273_305_F 2568 TKT_NC002163-TTATGAAGCGTGTTCTTTAGCAGGAC 661 TKT_NC002163-1569415-TCAAAACGCATTTTTACATCTTCGTTAA 946 1569415- TTCA 1569873_350_383_R AGGCTA1569873_255_284_F 2570 GLTA_NC002163- TCGTCTTTTTGATTCTTTCCCTGATA 381GLTANC002_163-1604930- TGTTCATGTTTAAATGATCAGGATAAAA 1347 1604930- ATGC1604529_109_142_R AGCACT 1604529_39_68_F 2571 TKT_NC002163-TGATCTTAAAAATTTCCGCCAACTTC 472 TKT_NC002163-1569415-TGCCATAGCAAAGCCTACAGCATT 1214 1569415- ATTC 1569903_139_162_R1569903_33_62_F 2572 TKT_NC002163- TAAGGTTTATTGTCTTTGTGGAGATG 164TKT_NC002163-1569415- TACATCTCCTTCGATAGAAATTTCATTG 886 1569415- GGGATTT1569903_313_345_R CTATC 1569903_207_239_F 2573 TKT_NC002163-TAGCCTTTAACGAAAATGTAAAAATG 213 TKT_NC002163-1569415-TAAGACAAGGTTTTGTGGATTTTTTAGC 865 1569415- CGTTTTGA 1569903_449_481_RTTGTT 1569903_350_383_F 2574 TKT_NC002163- TTCAAAAACTCCAGGCCATCCTGAAA665 TKT_NC002163-1569415- TTGCCATAGCAAAGCCTACAGCATT 1405 1569415-TTTCAAC 1569903_139_163_R 1569903_60_92_F 2575 GLTA_NC002163-TCGTCTTTTTGATTCTTTCCCTGATA 382 GLTA_NC002163-1604930-TGCCATTTCCATGTACTCTTCTCTAACA 1216 1604930- ATGCTC 1604529_139_168_R TT1604529_39_70_F 2576 GLYA_NC002163- TCAGCTATTTTTCCAGGTATCCAAGG 281GLYA_NC002_163-367572- ATTGCTTCTTACTTGCTTAGCATAAATT 756 367572- TGG368079_476_508_R TTCCA 368079_386_414_F 2577 GLYA_NC002163-TGGTGCGAGTGCTTATGCTCGTATTA 611 GLYA_NC002163-367572-TGCTCACCTGCTACAACAAGTCCAGCAA 1246 367572- T 368079_242_270_R T368079_148_174_F 2578 GLYA_NC002163- TGTAAGCTCTACAACCCACAAAACCT 622GLYA_NC002163-367572 - TTCCACCTTGGATACCTGGAAAAATAGC 1381 367572- TACG368079_384_416_R TGAAT 368079_298_327_F 2579 GLYA_NC002163-TGGTGGACATTTAACACATGGTGCAA 614 GLYA_NC002163-367572-TCAAGCTCTACACCATAAAAAAGCTCT 961 367572- A 368079_52_91_R CA368079_1_27_F 2580 PGM_NC002163- TGAGCAATGGGGCTTTGAAAGAATTT 455PGM_NC002163-327746- TTTGCTCTCCGCCAAAGTTTCCAC 1438 327746- TTAAAT328270_356_379_R 328270_254_285_F 2581 PGM_NC002163-TGAAAAGGGTGAAGTAGCAAATGGAG 425 PGK_NC002163-327746-TGCCCCATTGCTCATGATAGTAGCTAC 1219 327746- ATAG 328270_241_267_R328270_153_182_F 2582 PGM_NC002163- TGGCCTAATGGGCTTAATATCAATGA 568PGM_NC002163-327746- TGCACGCAAACGCTTTACTTCAGC 1200 327746- AAATTG328270_79_102_R 328270_19_50_F 2583 UNCA_NC002163-TAAGCATGCTGTGGCTTATCGTGAAA 160 UNCA_NC002163-112166-TGCCCTTTCTAAAAGTCTTGAGTGAAGA 1220 112186- TG 112647_196_225_R TA112647_114_141_F 2584 UNCA_NC002163- TGCTTCGGATCCAGCAGCACTTCAAT 532UNCA_NC0O2163-112166- TGCATGCTTACTCAAATCATCATAAACA 1206 112166- A112647_88_123_R ATTAAAGC 112647_3_29_F 2585 ASPA_NC002163-TTAATTTGCCAAAAATGCAACCAGGT 652 ASPA_NC002163-96692-TGCAAAAGTAACGGTTACATCTGCTCCA 1192 96692- AG 97166_403_432_R AT97166_308_335_F 2586 ASPA_NC002163- TCGCGTTGCAACAAAACTTTCTAAAG 370ASPA_NC002163-96692- TCATGATAGAACTACCTGGTTGCATTTT 991 96692- TATGT97166_916_346_R TGG 97166_228_258_F 2587 GLNA_NC002163-TGGAATGATGATAAAGATTTCGCAGA 547 GLNA_NC002163-658085-TGAGTTTGAACCATTTCAGAGCGAATAT 1176 658085- TAGCTA 657609_340_371_R CTAC657609_244_275_F 2588 TKT_NC002163- TCGCTACAGGCCCTTTAGGACAAG 371TKTNC002163-1569415- TCCCCATCTCCGCAAAGACAATAAA 1020 1569415-1569903_212_236_R 1569903_107_130_F 2589 TKT_NC002163-TGTTCTTTAGCAGGACTTCACAAACT 642 TKT_NC002163-1569415-TCCTTGTGCTTCAAAACGCATTTTTACA 1057 1569415- TGATAA 1569903_361_393_RTTTTC 1569903_265_296_F 2590 GLYA_NC002163- TGCCTATCTTTTTGCTGATATAGCAC505 GLYA_NC002163-367572- TCCTCTTGGGCCACGCAAAGTTTT 1047 367572- ATATTGC368095_917_340_R 368095_214_246_F 2591 GLYA_NC002163-TCCTTTGATGCATGTAATTGCTGCAA 353 GLYA_NC002163-367572-TCTTGAGCATTGGTTCTTACTTGTTTTG 1141 367572- AAGC 368095_485_516_R CATA368095_415_444_F 2592 PGM_NC002163_21_54_ TCCTAATGGACTTAATATCAATGAAA 332PGM_NC002163_116_142_R TCAAACGATCCGCATCACCATCAAAAG 949 F ATTGTGGA 2593PGM_NC002163_149_17 TAGATGAAAAAGGCGAAGTGGCTAAT 207 PGM_NC002163_247_277RTCCCCTTTAAAGCACCATTACTCATTAT 1023 6_F CC ACT 2594 GLNA_NC002163-TCTCCAACAAGCATACCAAAAAACC 633 GLNA_NC002163-658085-TCAAAAACAAACAATTCATTTTCTGGTC 945 658085- AA 657609_148_179_R CAAA657609_79_106_F 2595 ASPA_NC002163- TCCTCTTATTCCTGAACTACTTTTTC 347ASPA_NC002163-96685- TCAAGCTATATGCTACAACTCGTTC~ 960 96685- AACTTTCTTA97196_467_497_R AAC 97196_367_402_F ASPA_NC002163-TGCCCTAATCATACCTCAACATATAC 502 ASPA_NC002163-96685-TACAACCTTCCGATAATCAGGATCACAA 880 96685-97196_133_F AAAGACT97196_95_127_R TTAAT 2597 ASPA_NC002163- TGGAACACGAATTAATTCTCATCCTC 540ASPA_NC002163-96685- TAAGCTCCCGTATCTTGAGTCGCCTC 872 96685- ATTATCC97196_185_210_R 97196_85_117_F 2598 PGM_NC002163-TGGCAGCTAGAATAGTAGCTAAAATC 563 PGM_NC0021_63-327746-TCACGATCTAAATTTGGATAAGCCATAG 975 327746- CCTAC 328270_930_261_R GAAA328270_165_195_F 2599 PGM_NC002163- TGGGTCGTGGTTTTACAGAAAATTTC 593PGM_NC002163-327746- TTTTGCTCATGATCTGCATGAAGCATAA 1443 327746- TTATATATG328270_953_381_R A 328270_952_286_F 2600 PGM_NC002163-TGGGATGAAAAAGCGTTCTTTTATCC 577 PGM_NC002163-327746-TGATAAAAAGCACTAAGCGATGAAACAG 1178 327746- ATGA 328270_95_123_R C328270_1_30_F 2601 PGM_NC002163- TAAACACGGCTTTCCTATGGCTTATC 146PGM_NC002163-327746- TCAAGTGCTTTTACTTCTATAGGTTTAA 963 327746- CAAAT328270_314_345_R GGTG 328270_220_250_F 2602 UNCA_NC002163-TGTAGCTTATCGCGAAATGTCTTTGA 628 UNCA_NC002163-112166 -TGCTTGCTCTTTCAAGCAGTCTTGAATG 1258 112166- TTTT 112647_199_229_R AAG112647_123_152_F 2603 UNCA_NC002163- TCCAGATGGACAAATTTTCTTAGAAA 313UNCA_NC002163-112166- TCCGAAACTTGTTTTGTAGCTTTAATTT 1031 112166- CTGATTT112647_430_461_R GAGO 112647_333_965_F 2734 GYRA_AY291534_237_2TCACCCTCATGGTGATTCAGCTGTTT 265 GYRA_AY291534_268_288_RTTGCGCCATACGTACCATCGT 1407 64_F AT 2735 GYRA_AY291534_224_2TAATCGGTAAGTATCACCCTCATGGT 167 GYRA_AY291534_256_285_RTGCCATACGTACCATCGTTTCATAAACA 1213 52_F PAT PC 2736 GYRA_AY291534_170_1TAGGAATTACGGCTGATAAAGCGTAT 221 GYRA_AY291534_268_288_RTTGCGCCATACGTACCATCGT 1407 98_F AAA 2737 GYRA_AY291534_224_2TAATCGGTAAGTATCACCCTCATGT 167 GYRA_AY291534_319_346_RTATCGACAGATCCAAAGTTACCATGCCC 935 52_F PAT 2738 GYRA_NC002953-7005-TAAGGTATGACACCGGATAAATCATA 163 GYRA_NC002953-7005-TCTTGAGCCATACGTACCATTGC 1142 9668_166_195_F TAAA 9668965_287_R 2739GYRA_NC002953-7005- TAATGGGTAAATATCACCCTCATGGT 171 GYRA_NC002953-7005-TATCCATTGAACCAAAGGTACCTTGGCC 933 9668_221_249_F GAC 9668916_343_R 2740GYRA_NC002953-7005- TAATGGGTAAATATCACCCTCATGGT 171 GYRA_NC0029_53-7005-TAGCCATACGTACCATTGCTTCATAAAT 912 9668_221_249_F GAG 9668_253_283_R APAGYRA_NC002953-7005- TCACCCTCATGGTGACTCATCTATTT 264 GYRA_NC002953-7005-TCTTGAGCCATACGTACCATTGC 1142 9668_234_261_F AT 9668_265_287_R 2842CAPC_AF188935- TGGGATTATTGTTATCCTGTTATGCC 578 CAPC_AF188935-56074-TGGTAACCCTTGTCTTTGAATTGTATTT 1299 56074- ATTTPAPA 55628_348_378_R PCA55628_271_304_F 2843 CAPC_AF188935- TGATTATTGTTATCCTGTTATGCpCp 476CAPC_AF188935-56074- TGTAACCCTTGTCTTTGAATpTpGTATp 1314 56074- ATpTpTpPAG55628_349_377F_R TpTpGC 55628_273_303P_F 2844 CAPC_AF188935-TCCGTTGATTATTGTTATCCTGTTAT 331 CAPC_AF188935-56074-TGTTAATGGTAACCCTTGTGTTTGAATT 1344 56074- GCCATTTGAG 55628_349_384_RGTATTTGC 55628_268_303_F 2845 CAPC_AF188935- TCCGTTGATTATTGTTATCCTGTTAT331 CAPC_AF188935-56074- TAACCCTTGTCTTTGAATTGTATTTGCA 860 56074-GCCATTTGAG 55628_937_375_R ATTAATCCTGG 860 55628_268_303_F 2846PARC_X95819_33_58_F TCCAAAAAAATCAGCGCGTACAGTGG 302 PARC_X95819_121_153_RTAAAGGATAGCGGTAACTAAATGGCTGA 852 GCCAT 2847 PARC_X95819_65_92_FTACTTGGTAAATACCACCCACATGGT 199 PARC_X95819_157_178_RTACCCCAGTTCCCCTGACCTTC 889 GA 2848 PARC_X95819_69_93_FTGGTAAATACCACCCACATGGTGAC 596 PARC_X95819_97_128_RTGAGCCATGAGTACCATGGCTTCATAAC 1169 ATGC 2849 PARC_NC003997-TTCCGTAAGTCGGCTAAAACAGTCG 668 PARC_NC003997-3362578-TCCAAGTTTGACTTAAACGTACCATCGC 1001 3362578- 3365001_256_283_R3365001_181_205_F 2850 PARC_NC003997- TGTAACTATCACCCGCACGGTGAT 621PARC_NC003997-3362578- TCGTCAACACTACCATTATTACCATGCA 1099 3362578-3365001_304_335_R TCTC 3365001_217_240_F 2851 PARC_NC003997-TGTAACTATCACCCGCACGGTGAT 621 PARC_NC003997-3362578-TGACTTAAACGTACCATCGCTTCATATA 1162 3362578- 3365001_244_275_R CAGA3365001_217_240_F 2852 GYRA_AYE42140_- TAAATCTGCCCGTGTCGTTGGTGAC 150OYRA_AY642140_71_100_R TGCTAAAGTCTTGAGCCATACGAACAAT 1242 1_24_F GG 2853GYRA_AY642140_26_54_ TAATCGGTAAATATCACCCGCATGGT 166GYRA_AY642140_121_146_R TCGATCGAACCGAAGTTACCCTGACC 1069 _F GAC 2854GYRA_AY642140_26_54_ TARTCGGTAAATATCACCCGCATGGT 166GYRA_AY642140_58_89_R TGAGCCATACGAACAATGGTTTCATAAA 1168 F GAC CAGC 2860CYA_AF065404_348_1 TCCAACGAAGTACAATACARGACARA 305CYA_AF065404_1448_1472_R TCAGCTGTTAACGGCTTCAAGACCC 983 379_F AGAAGG 2861LEF_BA_AF065404_751_ TCGAAAGCTTTTGCATATTATATCGA 354LEF_BA_AF065404_843_881_R TCTTTAAGTTCTTCCAAGGATAGATTTA 1144 781_F GCCACTTTCTTGTTCG 2862 LEF_BA_AF065404_762_ TGCATATTATATCGAGCCACAGCATC 498LEF_BA_AF065404_843_881_R TCTTTAAGTTCTTCCAAGGATAGATTTA 1144 788_F GTTTCTTGTTCG 2917 MUTS_AY698802_106_1 TCCGCTGAATCTGTCGCCGC 326MUTS_AY698802_172_193_R TGCGGTCTGGCGCATATAGGTA 1237 25_F 2918MUTS_AY698802_172_1 TACCTATATGCGCCAGACCGC 187 MUTS_AY698802_228_252_RTCAATCTCGACTTTTTGTGCCGGTA 965 92_F 2919 MUTS_AY698802_228_2TACCGGCGCAAAAAGTCGAGATTGG 186 MUTS_AY698802_314_342_RTCGGTTTCAGTCATCTCCACCATAAAGG 1097 52_F T 2920 NUTS_AY698802_315_3TCTTTATGGTGGAGATGACTGAAACC 419 NUTS_AY698802_413_433_RTGCCAGCGACAGACCATCGTA 1210 42_F GA 2921 MUTS_AY698802_394_4TGGGCGTGGAACGTCCAC 585 MUTS_AY698802_497_519_R TCCGGTAACTGGGTCAGCTCGAA1040 11_F 2922 AB_MLST-11- TGGGCGATGCTGCgAAATGGTTAAAA 583 AB_MLST-11-TAGTATCACCACGTACACCCGGATCAGT 923 OIF007_991_1018_F GA OIF007_1110_1137_R2927 GAPA_NC002505_694_9 TCAATGAACGACCAACAAGTGATTGA 259GAPA_NC002505_29_58_R_1 TCCTTTATGCAACTTGGTATCARCAGGA 1060 21_F TG AT2928 GAPA_NC002505_694_7 TCGATGAACGACCAACAAGTGATTGA 361GAPA_NC002505_769_798_2_R TCCTTTATGCAACTTGGTATCAACCGGA 1061 21_2_F TG AT2929 GAPA_NC002505_694_7 TCGATGAACGACCAACAAGTGATTGA 361GAPA_NC002505_769_798_3_R TCCTTTATGCAACTTAGTATCAACCGGA 1059 21_2_F TG AT2932 INFB_EC_1364_1394_F TTGCTCGTGGTGCACAAGTAACGGAT 688INFB_EC_1439_1468_R TTGCTGCTTTCGCATGGTTAATCGCTTC 1410 ATTAC AA 2933INFB_EC_1364_1394_2_ TTGCTCGTGGTGCAIAAGTAACGGAT 689 INFB_EC_1439_1468_RTTGCTGCTTTCGCATGGTTAATCGCTTC 1410 F ATTAC AA 2934 INFB_EC_80_110_FTTGCCCGCGGTGCGGAAGTAACCGAT 685 INFB_EC_1439_1468_RTTGCTGCTTTCGCATGGTTAATCGCTTC 1410 ATTAC AA 2949 ACS_NC002516-TCGGCGCCTGCCTGATGA 376 ACS_NC002516-970624- TGGACCACGCCGAAGAACGG 1265970624- 971013_364_383_R 971013_299_316_F 2950 ARO_NC002516-26883-TCACCGTGCCGTTCAAGGAAGAG 267 ARO_NC002516-26883- TGTGTTGTCGCCGCGCAG 134127380_4_26_F 27380_111_128_R 2951 ARO_NC002516-26883-TTTCGAAGGGCCTTTCGACCTG 705 ARO_NC002516-26883-TCCTTGGCATACATCATGTCGTAGCA 1056 27380_356_377_F 27380_459_484_R 2952GUA_NC002516- TGGACTCCTCGGTGGTCGC 551 GUA_NC002516-4226546-TCGGCGAACATGGCCATCAC 1091 4226546- 4226174_127_146_R 4226174_23_41_F2953 GUA_NC002516- TGACCAGGTGATGGCCATGTTCG 448 GUA_NC002516-4226546-TGCTTCTCTTCCGGGTCGGC 1256 4226546- 4226174_214_233_R 4226174_120_142_F2954 GUA_NC002516- TTTTGAAGGTGATCCGTGCCAACG 710 GUA_NC002516-4226546-TGCTTGGTGGCTTCTTCGTCGAA 1259 4226546- 4226174_265_287_R4226174_155_178_F 2955 GUA_NC002516- TTCCTCGGCCGCCTGGC 670GUA_NC0102516-4226546- TGCGAGGAACTTCACGTCCTGC 1229 4226546-4226174_288_309_R 4226174_190_206_F 2956 GUA_NC002516-TCGGCCGCACCTTCATCGAAGT 374 GUA_NC002516-4226546- TCGTGGGCCTTGCCGGT 11114226546- 4226174_355_371_R 4226174_242_263_F 2957 MUT_NC002516-TGGAAGTCATCAAGCGCCTGGC 545 MUT_NC002516-5551158- TCACGGGCCAGCTCGTCT 9785551158- 5550717_99_116_R 5550717_5_26_F 2958 MUT_NC002516-TCGAGCAGCCGCTGCCG 358 MUT_NC002516-5551158- TCACCATGCGCCCGTTCACATA 9715551158- 5550717_256_277_R 5550717_152_168_F 2959 NUO_NC002516-TCAACCTCGGCCCGAACCA 249 NUO_NC002516-2984589- TCGGTGGTGGTAGCCGATCTC 10952984589- 2984954_97_117_R 2984954_8_26_F 2960 NUO_NC002516-TACTCTCGGTGGAGAAGCTCGC 195 NUO_NC002516-2984589-TTCAGGTACAGCAGGTGGTTCAGGAT 1376 2984589- 2984954_301_326_R2984954_218_239_F 3961 PPS_NC002516- TCCACGGTCATGGAGCGCTA 311PPS_NC002516-1915014- TCCATTTCCGACACGTCGTTGATCAC 1014 1915014-1915383_140_165_R 1915383_44_63_F 2962 PPS_NC002516- TCGCCATCGTCACCAACCG365 PPS_NC002516-1915014- TCCTGGCCATCCTGCAGGAT 1052 1915014-1915383_341_360_R 1915383_240_258_F 2963 TRP_NC002516-TGCTGGTACGGGTCGAGGA 527 TRP_NC002516-671831- TCGATCTCCTTGGCGTCCGA 1071671831- 672273_131 150_R 672273_24_42_F 2964 TRF_NC002516-TGCACATCGTGTCCAACGTCAC 490 TRP_NC002516-671831- TGATCTCCATGGCGCGGATCTT1182 671831- 672273_362_383_R 672273_261_282_F 2972 ABD_MLST-11-TGGGIGATGCTGCIAAATGGTTAAAA 592 AB_MLST-11- TAGTATCACCACGTACICCIGGATCAGT924 OIF007_1007_1034_F GA OIF007_126_1153_R 2993 OMPU_NC002505-TTCCCACCGATATCATGGCTTACCAC 667 OMPU_NC002505_544_567_RTCGGTCAGCAAAACGGTAGCTTGC 1094 674828- GG 675880_428_455_F 2994GAPA_NC002505- TCCTCAATGAACGAICAACAAGTGAT 335 GAPA_NC002505-506780-TTTTCCCTTTAAGCAACTTAGTATCAAC 1442 506780- TGATG 507937_769_802_R IGGAAT507937_691_721_F 2995 GAPA_NC002505- TCCTCIATGAACGAICAACAAGTGAT 339GAPA_NC002_505-506780- TCCATACCTTTATGCAACTTIGTATCAA 1008 506780- TGATG507937_769_803_R CIGGAAT 507937_691_721_2_F 2996 GAPA_NC002505-TCTCGATGAACGACCAACAAGTGATT 396 GAPA_NC002505-506780-TCGGAAATATTCTTTCAATACCTTTATG 1085 506780- GATG 507937_785_817_R CAACT507937_692_721_F 2997 GAPA_NC002505- TCCTCGATGAACGAICAACAAGTIAT 337GAPA_NC002505-506780- TCGGAAATATTCTTTCAATACCTTTATG 1085 506780- TGATG507937_785_817_R CAACT 507937_691_721_3_F 2998 GAPA_50002505-TCCTCAATGAATGATCAACAAGTGAT 336 GAPA_NC002505-506780-TCGGAAATATTCTTTCAATICCTTTITG 1087 506780- TGATG 507937_784_817_R CAACTT507937_691_721_4_F 2999 GAPA_NC002505- TCCTCIATGAAIGAICAACAAGTIAT 340GAPA_NC002505-506780- TCGGAAATATTCTTTCAATACCTTTATG 1086 506780- TGATG507937_784_817_2_R CAACTT 507937_691_721_5_F 3000 GAPA_NC002505-TCCTCGATGAATGAICAACAAGTIAT 338 PAPA_NC002505-506780-TTTGAATACCTTTATGCAACTTIGTATG 1430 506780- TGATG 507937_769_805_RAACIGGAAT 507937_691_721_6_F 3001 CTXB_NC002505-TCAGCATATGCACATGGAACACCTCA 275 CTXB_NC002505-1566967-TCCCGGCTAGAGATTCTGTATACGA 1026 1566967- 1567341_139_163_R1567341_46_71_F 3002 CTXB_NC002505- TCAGCATATGCACATGGAACACCTC 274CTXB_NC002505-1566967- TCCGGCTAGAGATTCTGTATAAAAT 1038 1566967-1567341_132_162_R ATG 1567341_46_70_F 3003 CTXB_NC002505-TCAGCATATGCACATGGAACACCTC 274 CTXB_NC002_505-1566967-TGCCGTATACGAAAATATCTTATCATTT 1225 1566967- 1567341_118_150_R AGGCT1567341_46_70_F 3004 TUFB_NC002758- TACAGGCCGTGTTGAACGTGG 180TUFB_NC002758-615038- TCAGCGTAGTCTAATAATTTACGGAACA 982 615038-616222_778_809_R TTTC 616222_684_704_F 3005 TUFB_50002758-TGCCGTGTTGAACGTGGTCAAAT 503 TUFB_NC002758-615038-TGCTTCAGCGTAGTCTAATAATTTACGG 1255 615038- 616222_783_813_R AAC616222_688_710_F 3006 TUFB_NC002758- TGTGGTCAAATCAAAGTTGGTGAAGA 638TUFB_NC002758-615038- TGCGTAGTCTAATAATTTACGGAACATT 1238 615038- A616222_778_807_R TC 616222_700_726_F 3007 TUFB_NC002758-TGGTCAAATCAAAGTTGGTGAAGAA 607 TUFB_NC002758-615038-TGCGTAGTCTAATAATTTACGGAACATT 1238 615038- 616222_778_807_R TC616222_702_726_F 3008 TUFB_NCC002758- TGAACGTGGTCAAATCAAAGTTGGTG 431TUFB_NC002758-615038- TCACCAGCTTCAGCGTAGTCTAATAATT 970 615038- AAGAA616222_785_818_R TACGGA 616222_696_726_F 3009 TUFB_NC002758-TCGTGTTGAACGTGGTCAAATCAAAG 386 TUFB_NC002758-615038-TCTTCAGCGTAGTCTAATAATTTACGGA 1134 615038- T 616222_778_812_R ACATTTC616222_690_716_F 3010 MECI-R_NC003923- TCACATATCGTGAGCAATGAACTC 261MECI-R_NC003923-41798- TGTGATATGGAGGTGTAGAAGGTG 1332 41798-41609_36_59_F41609_89_112_R 3011 MECI-R_NC003923- TGGGCGTGAGCAATGAACTGATTATA 584MECI-R_NC003923-41798- TGGCATGGAGGTGTAGAAGGTGTTATCA 128741798-41609_40_66_F C 41609_81_110_R TG 3012 MECI-R_140003923-TGGACACATATCGTGAGCAATGAACT 549 MECI-R_NC003923-41798-TGGGATGGAGGTGTAGAAGGTGTTATCA 1286 41798- GA 41609_81_110_R TG41609_33_60_2_F 3013 MECI-R_NC003923- TCGGTTTACAGATATCGTGAGCAATG 595MECI-R_NC003923-41798- TGGGGATATGGAGGTGTAGAAGGTGTTA 129041798-41609_29_60_F AACTGA 41609_81_113_R TGATG 3014 MUPR_X75439_2490_25TGGGCTCTTTCTCGCTTAAACACCT 587 MUPR_X75439_2548_2570_RTCTGCCTGCGGAAGTGAAATCGT 1130 14_F 3015 MUPR_X75439_2490_25TGGGCTCTTTCTCGCTTAAACACC 586 MUPR_X75439_2547_2568_RTGGCTGCGGAAGTGAAATCGTA 1281 13_F 3016 MUPR_X75439_2482_25TAGATAATTGGGCTCTTTCTCGCTTA 205 MUPR_X75439_2551_2573_RTAATCTGGCTGCGGAAGTGAAAT 876 10_F AAC 3017 MUPR_X75439_2490_25TGGGCTCTTTCTCGCTTAAACACCT 587 MUPR_X75439_2549_2573_RTAATCTGGCTGCGGAAGTGAAATCG 877 14_F 3018 MUPR_X75439_2482_25TAGATAATTGGGCTCTTTCTCGGTTA 205 MUPR_X75439_2559_2589_TGGTATATTCGTTAATTAATGTGGCTGC 1303 10_F AAC GGA 3019 MUPR_X75439_2490_25TGGGCTCTTTCTCGCTTAAAGACCT 587 MUPR_X75439_2554_2581_RTCGTTAATTAATCTGGCTGCGGAAAGTGA 1112 14_F 3020 AROE_NC003923-TGATGGCAAGTGGATAGGGTATAATA 474 AROE_NC003923-1674726-TAAGCAATACCTTTACTTGCACCACCT 868 1674726- CAG 1674277_309_335_R1674277_204_232_F 3021 AROE_NC003923- TGGCGAGTGGATAGGGTATAATACAG 570AROE_NC003923-1674726- TTCATAAGCAATACCTTTACTTGCAGCA 1378 1674726-1674277_311_339_R C 1674277_207_232_F 3022 AROE_NC003923-TGGCpAAGTpGGATpAGGGTpATpAA 572 AROE_NC003923-1674726-TAAGCAATACCpTpTpTpACTpTpGCpA 867 1674726- TpACpAG 1674277_311_335P_RCpCpAC 1674277_207_232P_F 3023 ARCC_NC003923- TCTGAAATGAATAGTGATAGAACTGT398 AROC_140003923-2725050- TCTTCTTCTTTCGTATAAAAAGGACCAA 1137 2725050-AGGCAC 2724595_214_245_R TTGG 2724595_124_155_F 3024 ARCC_NC003923-TGAATAGTGATAGAACTGTAGGCACA 437 ARCC_NC003923-2725050-TCTTCTTTCGTATAAAAAGGACCAATTG 1139 2725050- ATCGT 2724595_212_242_R GTT2724595_131_161_F 3025 ARCC_NC003923- TGAATAGTGATAGAACTGTAGGCACA 437ARCC_NC003923-2725050- TGCGCTAATTCTTCAACTTCTTCTTTCG 1232 2725050- ATCGT2724595_232_260_R T 2724595_131_161_F 3026 PTA_NC003923-TACAATGCTTGTTTATGCTGGTAAAG 177 PTA_NC003923-628885-TGTTCTTGATACACCTGGTTTCGTTTTG 1350 628885- CAG 629355_322_351_R AT629355_231_259_F 3027 PTA_NC003923- TACAATGCTTGTTTATGCTGGTAAAG 177PTA_NC003923-628885- TGGTACACCTGGTTTCGTTTTGATGATT 1301 628885- CAG629355_314_345_R TGTA 629355_231_259_F 3028 PTA_NC003923-TCTTGTTTATGCTGGTAAAGCAGATG 418 PTA_NC003923-628885-TCTTCTTGATACACCTGGTTTCGTTTTG 1350 628885- G 629355_322_351_R AT629355_237_263_F

Primer pair name codes and reference sequences are shown in Table 3. Theprimer name code typically represents the gene to which the given primerpair is targeted. The primer pair name may include specific coordinateswith respect to a reference sequence defined by an extraction of asection of sequence or defined by a GenBank gi number, or thecorresponding complementary sequence of the extraction, or the entireGenBank gi number as indicated by the label “no extraction.” Where “noextraction” is indicated for a reference sequence, the coordinates of aprimer pair named to the reference sequence are with respect to theGenBank gi listing. Gene abbreviations are shown in bold type in the“Gene Name” column.

To determine the exact primer hybridization coordinates of a given pairof primers on a given bioagent nucleic acid sequence and to determinethe sequences, molecular masses and base compositions of anamplification product to be obtained upon amplification of nucleic acidof a known bioagent with known sequence information in the region ofinterest with a given pair of primers, one with ordinary skill inbioinformatics is capable of obtaining alignments of the primers of thepresent invention with the GenBank gi number of the relevant nucleicacid sequence of the known bioagent. For example, the reference sequenceGenBank gi numbers (Table 3) provide the identities of the sequenceswhich can be obtained from GenBank. Alignments can be done using abioinformatics tool such as BLASTn provided to the public by NCBI(Bethesda, Md.). Alternatively, a relevant GenBank sequence may bedownloaded and imported into custom programmed or commercially availablebioinformatics programs wherein the alignment can be carried out todetermine the primer hybridization coordinates and the sequences,molecular masses and base compositions of the amplification product. Forexample, to obtain the hybridization coordinates of primer pair number2095 (SEQ ID NOs: 456:1261), First the forward primer (SEQ ID NO: 456)is subjected to a BLASTn search on the publicly available NCBI BLASTwebsite. “RefSeq_Genomic” is chosen as the BLAST database since the ginumbers refer to genomic sequences. The BLAST query is then performed.Among the top results returned is a match to GenBank gi number 21281729(Accession Number NC_(—)003923). The result shown below, indicates thatthe forward primer hybridizes to positions 1530282 . . . 1530307 of thegenomic sequence of Staphylococcus aureus subsp. aureus MW2 (representedby gi number 21281729). Staphylococcus aureus subsp. aureus MW2,complete genome Length = 2820462 Features in this part of subjectsequence: Panton-Valentine leukocidin chain F precursor Score = 52.0bits (26), Expect = 2e−05 Identities = 26/26 (100%), Gaps = 0/26 (0%)Strand = Plus/Plus Query 1 TGAGCTGCATCAACTGTATTGGATAG 26|||||||||||||||||||||||||| Sbjct 1530282 TGAGCTGCATCAACTGTATTGGATAG1530307

The hybridization coordinates of the reverse primer (SEQ ID NO: 1261)can be determined in a similar manner and thus, the bioagent identifyingamplicon can be defined in terms of genomic coordinates. Thequery/subject arrangement of the result would be presented inStrand=Plus/Minus format because the reverse strand hybridizes to thereverse complement of the genomic sequence. HThe preceding sequenceanalyses are well known to one with ordinary skill in bioinformatics andthus, Table 3 contains sufficient information to determine the primerhybridization coordinates of any of the primers of Table 2 to theapplicable reference sequences described therein. TABLE 3 Primer NameCodes and Reference Sequence Reference GenBank gi Primer name code GeneName Organism number 16S_EC 16S rRNA (16S ribosomal RNA gene)Escherichia coli 16127994 23S_EC 23S rRNA (23S ribosomal RNA gene)Escherichia coli 16127994 CAPC_BA capC (capsule biosynthesis gene)Bacillus anthracis 6470151 CYA_BA cya (cyclic AMP gene) Bacillusanthracis 4894216 DNAK_EC dnaK (chaperone dnaK gene) Escherichia coli16127994 GROL_EC groL (chaperonin groL) Escherichia coli 16127994HFLB_EC hflb (cell division protein peptidase Escherichia coli 16127994ftsH) INFB_EC infB (protein chain initiation factor Escherichia coli16127994 infB gene) LEF_BA lef (lethal factor) Bacillus anthracis21392688 PAG_BA pag (protective antigen) Bacillus anthracis 21392688RPLB_EC rplB (50S ribosomal protein L2) Escherichia coli 16127994RPOB_EC rpoB (DNA-directed RNA polymerase beta Escherichia coli 6127994chain) RPOC_EC rpoC (DNA-directed RNA polymerase Escherichia coli16127994 beta′ chain) SP101ET_SPET_11 Artificial Sequence ConcatenationArtificial 15674250 comprising: Sequence* - gki (glucose kinase) partialgene gtr (glutamine transporter protein) sequences of murI (glutamateracemase) Streptococcus mutS (DNA mismatch repair protein) pyogenes xpt(xanthine phosphoribosyl transferase) yqiL (acetyl-CoA-acetyltransferase) tkt (transketolase) SSPE_BA sspE (small acid-soluble sporeBacillus anthracis 30253828 protein) TUFB_EC tufB (Elongation factor Tu)Escherichia coli 16127994 VALS_EC valS (Valyl-tRNA synthetase)Escherichia coli 16127994 ASPS_EC aspS (Aspartyl-tRNA synthetase)Escherichia coli 16127994 CAF1_AF053947 caf1 (capsular protein caf1)Yersinia pestis 2996286 INV_U22457 inv (invasin) Yersinia pestis 1256565LL_NC003143 Y. pestis specific chromosomal genes - Yersinia pestis16120353 difference region BONTA_X52066 BoNT/A (neurotoxin type A)Clostridium 40381 botulinum MECA_Y14051 mecA methicillin resistance geneStaphylococcus 2791983 aureus TRPE_AY094355 trpE (anthranilate synthase(large Acinetobacter 20853695 component)) baumanii RECA_AF251469 recA(recombinase A) Acinetobacter 9965210 baumanii GYRA_AF100557 gyrA (DNAgyrase subunit A) Acinetobacter 4240540 baumanii GYRB_AB008700 gyrB (DNAgyrase subunit B) Acinetobacter 4514436 baumanii WAAA_Z96925 waaA(3-deoxy-D-manno-octulosonic-acid Acinetobacter 2765828 transferase)baumanii CJST_CJ Artificial Sequence Concatenation Artificial 15791399comprising: Sequence* - tkt (transketolase) partial gene glyA (serinehydroxymethyltransferase) sequences of gltA (citrate synthase)Campylobacter aspA (aspartate ammonia lyase) jejuni glnA (glutaminesynthase) pgm (phosphoglycerate mutase) uncA (ATP synthetase alphachain) RNASEP_BDP RNase P (ribonuclease P) Bordetella 33591275 pertussisRNASEP_BKM RNase P (ribonuclease P) Burkholderia 53723370 malleiRNASEP_BS RNase P (ribonuclease P) Bacillus subtilis 16077068 RNASEP_CLBRNase P (ribonuclease P) Clostridium 18308982 perfringens RNASEP_ECRNase P (ribonuclease P) Escherichia coli 16127994 RNASEP_RKP RNase P(ribonuclease P) Rickettsia 15603881 prowazekii RNASEP_SA RNase P(ribonuclease P) Staphylococcus 15922990 aureus RNASEP_VBC RNase P(ribonuclease P) Vibrio cholerae 15640032 ICD_CXB icd (isocitratedehydrogenase) Coxiella burnetii 29732244 IS1111A multi-locus IS1111Ainsertion element Acinetobacter 29732244 baumannii OMPA_AY485227 ompA(outer membrane protein A) Rickettsia 40287451 prowazekii OMPB_RKP ompB(outer membrane protein B) Rickettsia 15603881 prowazekii GLTA_RKP gltA(citrate synthase) Vibrio cholerae 15603881 TOXR_VBC toxR (transcriptionregulator toxR) Francisella 15640032 tularensis ASD_FRT asd (Aspartatesemialdehyde Francisella 56707187 dehydrogenase) tularensis GALE_FRTgalE (UDP-glucose 4-epimerase) Shigella flexneri 56707187 IPAH_SGF ipaH(invasion plasmid antigen) Campylobacter 30061571 jejuni HUPB_CJ hupB(DNA-binding protein Hu-beta) Coxiella burnetii 15791399 AB_MLSTArtificial Sequence Concatenation Artificial Sequenced comprising:Sequence* - in-house trpE (anthranilate synthase component partial gene(SEQ ID I)) sequences of NO: 1444) adk (adenylate kinase) AcinetobactermutY (adenine glycosylase) baumannii fumC (fumarate hydratase) efp(elongation factor p) ppa (pyrophosphate phospho- hydratase MUPR_X75439mupR (mupriocin resistance gene) Staphylococcus 438226 aureusPARC_X95819 parC (topoisomerase IV) Acinetobacter 1212748 baumanniiSED_M28521 sed (enterotoxin D) Staphylococcus 1492109 aureusPLA_AF053945 pla (plasminogen activator) Yersinia pestis 2996216SEJ_AF053140 sej (enterotoxin J) Staphylococcus 3372540 aureusGYRA_NC000912 gyrA (DNA gyrase subunit A) Mycoplasma 13507739 pneumoniaeACS_NC002516 acsA (Acetyl CoA Synthase) Pseudomonas 15595198 aeruginosaARO_NC002516 aroE (shikimate 5-dehydrogenase Pseudomonas 15595198aeruginosa GUA_NC002516 guaA (GMP synthase) Pseudomonas 15595198aeruginosa MUT_NC002516 mutL (DNA mismatch repair protein) Pseudomonas15595198 aeruginosa NUO_NC002516 nuoD (NADH dehydrogenase I chain C, D)Pseudomonas 15595198 aeruginosa PPS_NC002516 ppsA (Phosphoenolpyruvatesynthase) Pseudomonas 15595198 aeruginosa TRP_NC002516 trpE(Anthranilate synthetase Pseudomonas 15595198 component I) aeruginosaOMP2_NC000117 ompB (outer membrane protein B) Chlamydia 15604717trachomatis OMPA_NC000117 ompA (outer membrane protein B) Chlamydia15604717 trachomatis GYRA_NC000117 gyrA (DNA gyrase subunit A) Chlamydia15604717 trachomatis CTXA_NC002505 ctxA (Cholera toxin A subunit) Vibriocholerae 15640032 CTXB_NC002505 ctxB (Cholera toxin B subunit) Vibriocholerae 15640032 FUR_NC002505 fur (ferric uptake regulator protein)Vibrio cholerae 15640032 GAPA_NC_002505 gapA (glyceraldehyde-3-phosphateVibrio cholerae 15640032 dehydrogenase) GYRB_NC002505 gyrB (DNA gyrasesubunit B) Vibrio cholerae 15640032 OMPU_NC002505 ompU (outer membraneprotein) Vibrio cholerae 15640032 TCPA_NC002505 tcpA (toxin-coregulatedpilus) Vibrio cholerae 15640032 ASPA_NC002163 aspA (aspartate ammonialyase) Campylobacter 15791399 jejuni GLNA_NC002163 glnA (glutaminesynthetase) Campylobacter 15791399 jejuni GLTA_NC002163 gltA (glutamatesynthase) Campylobacter 15791399 jejuni GLYA_NC002163 glyA (serinehydroxymethyltransferase) Campylobacter 15791399 jejuni PGM_NC002163 pgm(phosphoglyceromutase) Campylobacter 15791399 jejuni TKT_NC002163 tkt(transketolase) Campylobacter 15791399 jejuni UNCA_NC002163 uncA (ATPsynthetase alpha chain) Campylobacter 15791399 jejuni AGR-III_NC003923agr-III (accessory gene regulator-III) Staphylococcus 21281729 aureusARCC_NC003923 arcC (carbamate kinase) Staphylococcus 21281729 aureusAROE_NC003923 aroE (shikimate 5-dehydrogenase Staphylococcus 21281729aureus BSA-A_NC003923 bsa-a (glutathione peroxidase) Staphylococcus21281729 aureus BSA-B_NC003923 bsa-b (epidermin biosynthesis proteinStaphylococcus 21281729 EpiB) aureus GLPF_NC003923 glpF (glyceroltransporter) Staphylococcus 21281729 aureus GMK_NC003923 gmk (guanylatekinase) Staphylococcus 21281729 aureus MECI-R_NC003923 mecR1 (truncatedmethicillin Staphylococcus 21281729 resistance protein) aureusPTA_NC003923 pta (phosphate acetyltransferase) Staphylococcus 21281729aureus PVLUK_NC003923 pvluk (Panton-Valentine leukocidin Staphylococcus21281729 chain F precursor) aureus SA442_NC003923 sa442 geneStaphylococcus 21281729 aureus SEA_NC003923 sea (staphylococcalenterotoxin A Staphylococcus 21281729 precursor) aureus SEC_NC003923sec4 (enterotoxin type C precursor) Staphylococcus 21281729 aureusTPI_NC003923 tpi (triosephosphate isomerase) Staphylococcus 21281729aureus YQI_NC003923 yqi (acetyl-CoA C-acetyltransferase Staphylococcus21281729 homologue) aureus GALE_AF513299 galE (galactose epimerase)Francisella 23506418 tularensis VVHA_NC004460 vVhA (cytotoxin, cytolysinprecursor) Vibrio vulnificus 27366463 TDH_NC004605 tdh (thermostabledirect hemolysin A) Vibrio 28899855 parahaemolyticus AGR-II_NC002745agr-II (accessory gene regulator-II) Staphylococcus 29165615 aureusPARC_NC003997 parC (topoisomerase IV) Bacillus anthracis 30260195GYRA_AY291534 gyrA (DNA gyrase subunit A) Bacillus anthracis 31323274AGR-I_AJ617706 agr-I (accessory gene regulator-I) Staphylococcus46019543 aureus AGR-IV_AJ617711 agr-IV (accessory gene regulator-III)Staphylococcus 46019563 aureus BLAZ_NC002952 blaZ (beta lactamase III)Staphylococcus 49482253 aureus ERMA_NC002952 ermA (rRNAmethyltransferase A) Staphylococcus 49482253 aureus ERMB_Y13600 ermB(rRNA methyltransferase B) Staphylococcus 49482253 aureusSEA-SEE_NC002952 sea (staphylococcal enterotoxin A Staphylococcus49482253 precursor) aureus SEA-SEE_NC002952 sea (staphylococcalenterotoxin A Staphylococcus 49482253 precursor) aureus SEE_NC002952 sea(staphylococcal enterotoxin A Staphylococcus 49482253 precursor) aureusSEH_NC002953 seh (staphylococcal enterotoxin H) Staphylococcus 49484912aureus ERMC_NC005908 ermC (rRNA methyltransferase C) Staphylococcus49489772 aureus MUTS_AY698802 mutS (DNA mismatch repair protein)Shigella boydii 52698233 NUC_NC002758 nuc (staphylococcal nuclease)Staphylococcus 57634611 aureus SEB_NC002758 seb (enterotoxin type Bprecursor) Staphylococcus 57634611 aureus SEG_NC002758 seg(staphylococcal enterotoxin G) Staphylococcus 57634611 aureusSEI_NC002758 sei (staphylococcal enterotoxin I) Staphylococcus 57634611aureus TSST_NC002758 tsst (toxic shock syndrome toxin-1) Staphylococcus57634611 aureus TUFB_NC002758 tufB (Elongation factor Tu) Staphylococcus57634611 aureus

Note: artificial reference sequences represent concatenations of partialgene extractions from the indicated reference gi number. Partialsequences were used to create the concatenated sequence because completegene sequences were not necessary for primer design.

Example 2 Sample Preparation and PCR

Genomic DNA was prepared from samples using the DNeasy Tissue Kit(Qiagen, Valencia, Calif.) according to the manufacturer's protocols.

All PCR reactions were assembled in 50 μL reaction volumes in a 96-wellmicrotiter plate format using a Packard MPII liquid handling roboticplatform and M. J. Dyad thermocyclers (MJ research, Waltham, Mass.) orEppendorf Mastercycler thermocyclers (Eppendorf, Westbury, N.Y.). ThePCR reaction mixture consisted of 4 units of Amplitaq Gold, 1× buffer II(Applied Biosystems, Foster City, Calif.), 1.5 mM MgCl₂, 0.4 M betaine,800 μM dNTP mixture and 250 nM of each primer. The following typical PCRconditions were used: 95° C. for 10 min followed by 8 cycles of 95° C.for 30 seconds, 48° C. for 30 seconds, and 72° C. 30 seconds with the48° C. annealing temperature increasing 0.9° C. with each of the eightcycles. The PCR was then continued for 37 additional cycles of 95° C.for 15 seconds, 56° C. for 20 seconds, and 72° C. 20 seconds.

Example 3 Purification of PCR Products for Mass Spectrometry with IonExchange Resin-Magnetic Beads

For solution capture of nucleic acids with ion exchange resin linked tomagnetic beads, 25 μl of a 2.5 mg/mL suspension of BioClone amineterminated superparamagnetic beads were added to 25 to 50 μl of a PCR(or RT-PCR) reaction containing approximately 10 pM of a typical PCRamplification product. The above suspension was mixed for approximately5 minutes by vortexing or pipetting, after which the liquid was removedafter using a magnetic separator. The beads containing bound PCRamplification product were then washed three times with 50 mM ammoniumbicarbonate/50% MeOH or 100 mM ammonium bicarbonate/50% MeOH, followedby three more washes with 50% MeOH. The bound PCR amplicon was elutedwith a solution of 25 mM piperidine, 25 mM imidazole, 35% MeOH whichincluded peptide calibration standards.

Example 4 Mass Spectrometry and Base Composition Analysis

The ESI-FTICR mass spectrometer is based on a Bruker Daltonics(Billerica, Mass.) Apex II 70e electrospray ionization Fourier transformion cyclotron resonance mass spectrometer that employs an activelyshielded 7 Tesla superconducting magnet. The active shielding constrainsthe majority of the fringing magnetic field from the superconductingmagnet to a relatively small volume. Thus, components that might beadversely affected by stray magnetic fields, such as CRT monitors,robotic components, and other electronics, can operate in closeproximity to the FTICR spectrometer. All aspects of pulse sequencecontrol and data acquisition were performed on a 600 MHz Pentium II datastation running Bruker's Xmass software under Windows NT 4.0 operatingsystem. Sample aliquots, typically 15 μl, were extracted directly from96-well microtiter plates using a CTC HTS PAL autosampler (LEAPTechnologies, Carrboro, N.C.) triggered by the FTICR data station.Samples were injected directly into a 10 μl sample loop integrated witha fluidics handling system that supplies the 100 μl/hr flow rate to theESI source. Ions were formed via electrospray ionization in a modifiedAnalytica (Branford, Conn.) source employing an off axis, groundedelectrospray probe positioned approximately 1.5 cm from the metalizedterminus of a glass desolvation capillary. The atmospheric pressure endof the glass capillary was biased at 6000 V relative to the ESI needleduring data acquisition. A counter-current flow of dry N₂ was employedto assist in the desolvation process. Ions were accumulated in anexternal ion reservoir comprised of an rf-only hexapole, a skimmer cone,and an auxiliary gate electrode, prior to injection into the trapped ioncell where they were mass analyzed. Ionization duty cycles greater than99% were achieved by simultaneously accumulating ions in the externalion reservoir during ion detection. Each detection event consisted of 1Mdata points digitized over 2.3 s. To improve the signal-to-noise ratio(S/N), 32 scans were co-added for a total data acquisition time of 74 s.

The ESI-TOF mass spectrometer is based on a Bruker Daltonics MicroTOF™.Ions from the ESI source undergo orthogonal ion extraction and arefocused in a reflectron prior to detection. The TOF and FTICR areequipped with the same automated sample handling and fluidics describedabove. Ions are formed in the standard MicroTOF™ ESI source that isequipped with the same off-axis sprayer and glass capillary as the FTICRESI source. Consequently, source conditions were the same as thosedescribed above. External ion accumulation was also employed to improveionization duty cycle during data acquisition. Each detection event onthe TOF was comprised of 75,000 data points digitized over 75 μs.

The sample delivery scheme allows sample aliquots to be rapidly injectedinto the electrospray source at high flow rate and subsequently beelectrosprayed at a much lower flow rate for improved ESI sensitivity.Prior to injecting a sample, a bolus of buffer was injected at a highflow rate to rinse the transfer line and spray needle to avoid samplecontamination/carryover. Following the rinse step, the autosamplerinjected the next sample and the flow rate was switched to low flow.Following a brief equilibration delay, data acquisition commenced. Asspectra were co-added, the autosampler continued rinsing the syringe andpicking up buffer to rinse the injector and sample transfer line. Ingeneral, two syringe rinses and one injector rinse were required tominimize sample carryover. During a routine screening protocol a newsample mixture was injected every 106 seconds. More recently a fast washstation for the syringe needle has been implemented which, when combinedwith shorter acquisition times, facilitates the acquisition of massspectra at a rate of just under one spectrum/minute.

Raw mass spectra were post-calibrated with an internal mass standard anddeconvoluted to monoisotopic molecular masses. Unambiguous basecompositions were derived from the exact mass measurements of thecomplementary single-stranded oligonucleotides. Quantitative results areobtained by comparing the peak heights with an internal PCR calibrationstandard present in every PCR well at 500 molecules per well.Calibration methods are commonly owned and disclosed in U.S. ProvisionalPatent Application Ser. No. 60/545,425 which is incorporated herein byreference in entirety.

Example 5 De Novo Determination of Base Composition of AmplificationProducts Using Molecular Mass Modified Deoxynucleotide Triphosphates

Because the molecular masses of the four natural nucleobases have arelatively narrow molecular mass range (A=313.058, G=329.052, C=289.046,T=304.046—See Table 4), a persistent source of ambiguity in assignmentof base composition can occur as follows: two nucleic acid strandshaving different base composition may have a difference of about 1 Dawhen the base composition difference between the two strands is G

A (−15.994) combined with C

T (+15.000). For example, one 99-mer nucleic acid strand having a basecomposition of A₂₇G₃₀C₂₁T₂₁ has a theoretical molecular mass of30779.058 while another 99-mer nucleic acid strand having a basecomposition of A₂₆G₃₁C₂₂T₂₀ has a theoretical molecular mass of30780.052. A 1 Da difference in molecular mass may be within theexperimental error of a molecular mass measurement and thus, therelatively narrow molecular mass range of the four natural nucleobasesimposes an uncertainty factor.

The present invention provides for a means for removing this theoretical1 Da uncertainty factor through amplification of a nucleic acid with onemass-tagged nucleobase and three natural nucleobases. The term“nucleobase” as used herein is synonymous with other terms in use in theart including “nucleotide,” “deoxynucleotide,” “nucleotide residue,”“deoxynucleotide residue,” “nucleotide triphosphate (NTP),” ordeoxynucleotide triphosphate (dNTP).

Addition of significant mass to one of the 4 nucleobases (dNTPs) in anamplification reaction, or in the primers themselves, will result in asignificant difference in mass of the resulting amplification product(significantly greater than 1 Da) arising from ambiguities arising fromthe G

A combined with C

T event (Table 4). Thus, the same the G

A (−15.994) event combined with 5-Iodo-C

T (−110.900) event would result in a molecular mass difference of126.894. If the molecular mass of the base composition A₂₇G₃₀5-Iodo-C₂₁T₂₁ (33422.958) is compared with A₂₆G3,5-Iodo-C₂₂T₂₀,(33549.852) the theoretical molecular mass difference is +126.894. Theexperimental error of a molecular mass measurement is not significantwith regard to this molecular mass difference. Furthermore, the onlybase composition consistent with a measured molecular mass of the 99-mernucleic acid is A₂₇G₃₀5-Iodo-C₂₁T₂₁. In contrast, the analogousamplification without the mass tag has 18 possible base compositions.TABLE 4 Molecular Masses of Natural Nucleobases and the Mass-ModifiedNucleobase 5-Iodo-C and Molecular Mass Differences Resulting fromTransitions Nucleobase Molecular Mass Transition Molecular Mass A313.058 A-->T −9.012 A 313.058 A-->C −24.012 A 313.058 A-->5-Iodo-C101.888 A 313.058 A-->G 15.994 T 304.046 T-->A 9.012 T 304.046 T-->C−15.000 T 304.046 T-->5-Iodo-C 110.900 T 304.046 T-->G 25.006 C 289.046C-->A 24.012 C 289.046 C-->T 15.000 C 289.046 C-->G 40.006 5-Iodo-C414.946 5-Iodo-C-->A −101.888 5-Iodo-C 414.946 5-Iodo-C-->T −110.9005-Iodo-C 414.946 5-Iodo-C-->G −85.894 G 329.052 G-->A −15.994 G 329.052G-->T −25.006 G 329.052 G-->C −40.006 G 329.052 G-->5-Iodo-C 85.894

Mass spectra of bioagent-identifying amplicons were analyzedindependently using a maximum-likelihood processor, such as is widelyused in radar signal processing. This processor, referred to as GenX,first makes maximum likelihood estimates of the input to the massspectrometer for each primer by running matched filters for each basecomposition aggregate on the input data. This includes the GenX responseto a calibrant for each primer.

The algorithm emphasizes performance predictions culminating inprobability-of-detection versus probability-of-false-alarm plots forconditions involving complex backgrounds of naturally occurringorganisms and environmental contaminants. Matched filters consist of apriori expectations of signal values given the set of primers used foreach of the bioagents. A genomic sequence database is used to define themass base count matched filters. The database contains the sequences ofknown bacterial bioagents and includes threat organisms as well asbenign background organisms. The latter is used to estimate and subtractthe spectral signature produced by the background organisms. A maximumlikelihood detection of known background organisms is implemented usingmatched filters and a running-sum estimate of the noise covariance.Background signal strengths are estimated and used along with thematched filters to form signatures which are then subtracted. Themaximum likelihood process is applied to this “cleaned up” data in asimilar manner employing matched filters for the organisms and arunning-sum estimate of the noise-covariance for the cleaned up data.

The amplitudes of all base compositions of bioagent-identifyingamplicons for each primer are calibrated and a final maximum likelihoodamplitude estimate per organism is made based upon the multiple singleprimer estimates. Models of all system noise are factored into thistwo-stage maximum likelihood calculation. The processor reports thenumber of molecules of each base composition contained in the spectra.The quantity of amplification product corresponding to the appropriateprimer set is reported as well as the quantities of primers remainingupon completion of the amplification reaction.

Base count blurring can be carried out as follows. “Electronic PCR” canbe conducted on nucleotide sequences of the desired bioagents to obtainthe different expected base counts that could be obtained for eachprimer pair. See for example, ncbi.nlm.nih.gov/sutils/e-pcr/; Schuler,Genome Res. 7:541-50, 1997. In one illustrative embodiment, one or morespreadsheets, such as Microsoft Excel workbooks contain a plurality ofworksheets. First in this example, there is a worksheet with a namesimilar to the workbook name; this worksheet contains the raw electronicPCR data. Second, there is a worksheet named “filtered bioagents basecount” that contains bioagent name and base count; there is a separaterecord for each strain after removing sequences that are not identifiedwith a genus and species and removing all sequences for bioagents withless than 10 strains. Third, there is a worksheet, “Sheet1” thatcontains the frequency of substitutions, insertions, or deletions forthis primer pair. This data is generated by first creating a pivot tablefrom the data in the “filtered bioagents base count” worksheet and thenexecuting an Excel VBA macro. The macro creates a table of differencesin base counts for bioagents of the same species, but different strains.One of ordinary skill in the art may understand additional pathways forobtaining similar table differences without undo experimentation.

Application of an exemplary script, involves the user defining athreshold that specifies the fraction of the strains that arerepresented by the reference set of base counts for each bioagent. Thereference set of base counts for each bioagent may contain as manydifferent base counts as are needed to meet or exceed the threshold. Theset of reference base counts is defined by taking the most abundantstrain's base type composition and adding it to the reference set andthen the next most abundant strain's base type composition is addeduntil the threshold is met or exceeded. The current set of data wasobtained using a threshold of 55%, which was obtained empirically.

For each base count not included in the reference base count set forthat bioagent, the script then proceeds to determine the manner in whichthe current base count differs from each of the base counts in thereference set. This difference may be represented as a combination ofsubstitutions, Si=Xi, and insertions, Ii=Yi, or deletions, Di=Zi. Ifthere is more than one reference base count, then the reporteddifference is chosen using rules that aim to minimize the number ofchanges and, in instances with the same number of changes, minimize thenumber of insertions or deletions. Therefore, the primary rule is toidentify the difference with the minimum sum (Xi+Yi) or (Xi+Zi), e.g.,one insertion rather than two substitutions. If there are two or moredifferences with the minimum sum, then the one that will be reported isthe one that contains the most substitutions.

Differences between a base count and a reference composition arecategorized as one, two, or more substitutions, one, two, or moreinsertions, one, two, or more deletions, and combinations ofsubstitutions and insertions or deletions. The different classes ofnucleobase changes and their probabilities of occurrence have beendelineated in U.S. Patent Application Publication No. 2004209260 (U.S.application Ser. No. 10/418,514) which is incorporated herein byreference in entirety.

Example 6 Use of Broad Range Survey and Division Wide Primer Pairs forIdentification of Bacteria in an Epidemic Surveillance Investigation

This investigation employed a set of 16 primer pairs which is hereindesignated the “surveillance primer set” and comprises broad rangesurvey primer pairs, division wide primer pairs and a single Bacillusclade primer pair. The surveillance primer set is shown in Table 5 andconsists of primer pairs originally listed in Table 2. This surveillanceset comprises primers with T modifications (note TMOD designation inprimer names) which constitutes a functional improvement with regard toprevention of non-templated adenylation (vide supra) relative tooriginally selected primers which are displayed below in the same row.Primer pair 449 (non-T modified) has been modified twice. Itspredecessors are primer pairs 70 and 357, displayed below in the samerow. Primer pair 360 has also been modified twice and its predecessorsare primer pairs 17 and 118. TABLE 5 Bacterial Primer Pairs of theSurveillance Primer Set Forward Reverse Primer Primer Primer Pair (SEQID (SEQ ID No. Forward Primer Name NO:) Reverse Primer Name NO:) TargetGene 346 16S_EC_713_732_TMOD_F 202 16S_EC_789_809_TMOD_R 1110 16S rRNA10 16S_EC_713_732_F 21 16S_EC_789_809 798 16S rRNA 34716S_EC_785_806_TMOD_F 560 16S_EC_880_897_TMOD_R 1278 16S rRNA 1116S_EC_785_806_F 118 16S_EC_880_897_R 830 16S rRNA 34816S_EC_960_981_TMOD_F 706 16S_EC_1054_1073_TMOD_R 895 16S rRNA 1416S_EC_960_981_F 672 16S_EC_1054_1073_R 735 16S rRNA 34923S_EC_1826_1843_TMOD_F 401 23S_EC_1906_1924_TMOD_R 1156 23S rRNA 1623S_EC_1826_1843_F 80 23S_EC_1906_1924_R 805 23S rRNA 352INFB_EC_1365_1393_TMOD_F 687 INFB_EC_1439_1467_TMOD_R 1411 infB 34INFB_EC_1365_1393_F 524 INFB_EC_1439_1467_R 1248 infB 354RPOC_EC_2218_2241_TMOD_F 405 RPOC_EC_2313_2337_TMOD_R 1072 rpoC 52RPOC_EC_2218_2241_F 81 RPOC_EC_2313_2337_R 790 rpoC 355SSPE_BA_115_137_TMOD_F 255 SSPE_BA_197_222_TMOD_R 1402 sspE 58SSPE_BA_115_137_F 45 SSPE_BA_197_222_R 1201 sspE 356RPLB_EC_650_679_TMOD_F 232 RPLB_EC_739_762_TMOD_R 592 rplB 66RPLB_EC_650_679_F 98 RPLB_EC_739_762_R 999 rplB 358VALS_EC_1105_1124_TMOD_F 385 VALS_EC_1195_1218_TMOD_R 1093 valS 71VALS_EC_1105_1124_F 77 VALS_EC_1195_1218_R 795 valS 359RPOB_EC_1845_1866_TMOD_F 659 RPOB_EC_1909_1929_TMOD_R 1250 rpoB 72RPOB_EC_1845_1866_F 233 RPOB_EC_1909_1929_R 825 rpoB 36023S_EC_2646_2667_TMOD_F 409 23S_EC_2745_2765_TMOD_R 1434 23S rRNA 11823S_EC_2646_2667_F 84 23S_EC_2745_2765_R 1389 23S rRNA 1723S_EC_2645_2669_F 408 23S_EC_2744_2761_R 1252 23S rRNA 36116S_EC_1090_1111_2_TMOD_F 697 16S_EC_1175_1196_TMOD_R 1398 16S rRNA 316S_EC_1090_1111_2_F 651 16S_EC_1175_1196_R 1159 16S rRNA 362RPOB_EC_3799_3821_TMOD_F 581 RPOB_EC_3862_3888_TMOD_R 1325 rpoB 289RPOB_EC_3799_3821_F 124 RPOB_EC_3862_3888_R 840 rpoB 363RPOC_EC_2146_2174_TMOD_F 284 RPOC_EC_2227_2245_TMOD_R 898 rpoC 290RPOC_EC_2146_2174_F 52 RPOC_EC_2227_2245_R 736 rpoC 367TUFB_EC_957_979_TMOD_F 308 TUFB_EC_1034_1058_TMOD_R 1276 tufB 293TUFB_EC_957_979_F 55 TUFB_EC_1034_1058_R 829 tufB 449 RPLB_EC_690_710_F309 RPLB_EC_737_758_R 1336 rplB 357 RPLB_EC_688_710_TMOD_F 296RPLB_EC_736_757_TMOD_R 1337 rplB 67 RPLB_EC_688_710_F 54RPLB_EC_736_757_R 842 rplB

The 16 primer pairs of the surveillance set are used to produce bioagentidentifying amplicons whose base compositions are sufficiently differentamongst all known bacteria at the species level to identify, at areasonable confidence level, any given bacterium at the species level.As shown in Tables 6A-E, common respiratory bacterial pathogens can bedistinguished by the base compositions of bioagent identifying ampliconsobtained using the 16 primer pairs of the surveillance set. In somecases, triangulation identification improves the confidence level forspecies assignment. For example, nucleic acid from Streptococcuspyogenes can be amplified by nine of the sixteen surveillance primerpairs and Streptococcus pneumoniae can be amplified by ten of thesixteen surveillance primer pairs. The base compositions of the bioagentidentifying amplicons are identical for only one of the analogousbioagent identifying amplicons and differ in all of the remaininganalogous bioagent identifying amplicons by up to four bases perbioagent identifying amplicon. The resolving power of the surveillanceset was confirmed by determination of base compositions for 120 isolatesof respiratory pathogens representing 70 different bacterial species andthe results indicated that natural variations (usually only one or twobase substitutions per bioagent identifying amplicon) amongst multipleisolates of the same species did not prevent correct identification ofmajor pathogenic organisms at the species level.

Bacillus anthracis is a well known biological warfare agent which hasemerged in domestic terrorism in recent years. Since it was envisionedto produce bioagent identifying amplicons for identification of Bacillusanthracis, additional drill-down analysis primers were designed totarget genes present on virulence plasmids of Bacillus anthracis so thatadditional confidence could be reached in positive identification ofthis pathogenic organism. Three drill-down analysis primers weredesigned and are listed in Tables 2 and 6. In Table 6, the drill-downset comprises primers with T modifications (note TMOD designation inprimer names) which constitutes a functional improvement with regard toprevention of non-templated adenylation (vide supra) relative tooriginally selected primers which are displayed below in the same row.TABLE 6 Drill-Down Primer Pairs for Confirmation of Identification ofBacillus anthracis Forward Reverse Primer Primer Primer Pair (SEQ ID(SEQ ID No. Forward Primer Name NO:) Reverse Primer Name NO:) TargetGene 350 CAPC_BA_274_303_TMOD_F 476 CAPC_BA_349_376_TMOD_R 1314 capC 24CAPC_BA_274_303_F 109 CAPC_BA_349_376_R 837 capC 351CYA_BA_1353_1379_TMOD_F 355 CYA_BA_1448_1467_TMOD_R 1423 cyA 30CYA_BA_1353_1379_F 64 CYA_BA_1448_1467_R 1342 cyA 353LEF_BA_756_781_TMOD_F 220 LEF_BA_843_872_TMOD_R 1394 lef 37LEF_BA_756_781_F 26 LEF_BA_843_872_R 1135 lef

Phylogenetic coverage of bacterial space of the sixteen surveillanceprimers of Table 5 and the three Bacillus anthracis drill-down primersof Table 6 is shown in FIG. 3 which lists common pathogenic bacteria.FIG. 3 is not meant to be comprehensive in illustrating all speciesidentified by the primers. Only pathogenic bacteria are listed asrepresentative examples of the bacterial species that can be identifiedby the primers and methods of the present invention. Nucleic acid ofgroups of bacteria enclosed within the polygons of FIG. 3 can beamplified to obtain bioagent identifying amplicons using the primer pairnumbers listed in the upper right hand corner of each polygon. Primercoverage for polygons within polygons is additive. As an illustrativeexample, bioagent identifying amplicons can be obtained for Chlamydiatrachomatis by amplification with, for example, primer pairs 346-349,360 and 361, but not with any of the remaining primers of thesurveillance primer set. On the other hand, bioagent identifyingamplicons can be obtained from nucleic acid originating from Bacillusanthracis (located within 5 successive polygons) using, for example, anyof the following primer pairs: 346-349, 360, 361 (base polygon), 356,449 (second polygon), 352 (third polygon), 355 (fourth polygon), 350,351 and 353 (fifth polygon). Multiple coverage of a given organism withmultiple primers provides for increased confidence level inidentification of the organism as a result of enabling broadtriangulation identification.

In Tables 7A-E, base compositions of respiratory pathogens for primertarget regions are shown. Two entries in a cell, represent variation inribosomal DNA operons. The most predominant base composition is shownfirst and the minor (frequently a single operon) is indicated by anasterisk (*). Entries with NO DATA mean that the primer would not beexpected to prime this species due to mismatches between the primer andtarget region, as determined by theoretical PCR. TABLE 7A BaseCompositions of Common Respiratory Pathogens for Bioagent IdentifyingAmplicons Corresponding to Primer Pair Nos: 346, 347 and 348 Primer 346Primer 347 Primer 348 Organism Strain [A G C T] [A G C T] [A G C T]Klebsiella MGH78578 [29 32 25 13] [23 38 28 26] [26 32 28 30] pneumoniae[29 31 25 13]* [23 37 28 26]* [26 31 28 30]* Yersinia pestis CO-92Biovar [29 32 25 13] [22 39 28 26] [29 30 28 29] Orientalis [30 30 2729]* Yersinia pestis KIM5 P12 (Biovar [29 32 25 13] [22 39 28 26] [29 3028 29] Mediaevalis) Yersinia pestis 91001 [29 32 25 13] [22 39 28 26][29 30 28 29] [30 30 27 29]* Haemophilus KW20 [28 31 23 17] [24 37 2527] [29 30 28 29] influenzae Pseudomonas PAO1 [30 31 23 15] [26 36 2924] [26 32 29 29] aeruginosa [27 36 29 23]* Pseudomonas Pf0-1 [30 31 2315] [26 35 29 25] [28 31 28 29] fluorescens Pseudomonas KT2440 [30 31 2315] [28 33 27 27] [27 32 29 28] putida Legionella Philadelphia-1 [30 3024 15] [33 33 23 27] [29 28 28 31] pneumophila Francisella schu 4 [32 2922 16] [28 38 26 26] [25 32 28 31] tularensis Bordetella Tohama I [30 2924 16] [23 37 30 24] [30 32 30 26] pertussis Burkholderia J2315 [29 2927 14] [27 32 26 29] [27 36 31 24] cepacia [20 42 35 19]* BurkholderiaK96243 [29 29 27 14] [27 32 26 29] [27 36 31 24] pseudomallei NeisseriaFA 1090, ATCC [29 28 24 18] [27 34 26 28] [24 36 29 27] gonorrhoeae700825 Neisseria MC58 (serogroup B) [29 28 26 16] [27 34 27 27] [25 3530 26] meningitidis Neisseria serogroup C, FAM18 [29 28 26 16] [27 34 2727] [25 35 30 26] meningitidis Neisseria Z2491 (serogroup A) [29 28 2616] [27 34 27 27] [25 35 30 26] meningitidis Chlamydophila TW-183 [31 2722 19] NO DATA [32 27 27 29] pneumoniae Chlamydophila AR39 [31 27 22 19]NO DATA [32 27 27 29] pneumoniae Chlamydophila CWL029 [31 27 22 19] NODATA [32 27 27 29] pneumoniae Chlamydophila J138 [31 27 22 19] NO DATA[32 27 27 29] pneumoniae Corynebacterium NCTC13129 [29 34 21 15] [22 3831 25] [22 33 25 34] diphtheriae Mycobacterium k10 [27 36 21 15] [22 3730 28] [21 36 27 30] avium Mycobacterium 104 [27 36 21 15] [22 37 30 28][21 36 27 30] avium Mycobacterium CSU#93 [27 36 21 15] [22 37 30 28] [2136 27 30] tuberculosis Mycobacterium CDC 1551 [27 36 21 15] [22 37 3028] [21 36 27 30] tuberculosis Mycobacterium H37Rv (lab strain) [27 3621 15] [22 37 30 28] [21 36 27 30] tuberculosis Mycoplasma M129 [31 2919 20] NO DATA NO DATA pneumoniae Staphylococcus MRSA252 [27 30 21 21][25 35 30 26] [30 29 30 29] aureus [29 31 30 29]* Staphylococcus MSSA476[27 30 21 21] [25 35 30 26] [30 29 30 29] aureus [30 29 29 30]*Staphylococcus COL [27 30 21 21] [25 35 30 26] [30 29 30 29] aureus [3029 29 30]* Staphylococcus Mu50 [27 30 21 21] [25 35 30 26] [30 29 30 29]aureus [30 29 29 30]* Staphylococcus MW2 [27 30 21 21] [25 35 30 26] [3029 30 29] aureus [30 29 29 30]* Staphylococcus N315 [27 30 21 21] [25 3530 26] [30 29 30 29] aureus [30 29 29 30]* Staphylococcus NCTC 8325 [2730 21 21] [25 35 30 26] [30 29 30 29] aureus [25 35 31 26]* [30 29 2930] Streptococcus NEM316 [26 32 23 18] [24 36 31 25] [25 32 29 30]agalactiae [24 36 30 26]* Streptococcus NC_002955 [26 32 23 18] [23 3731 25] [29 30 25 32] equi Streptococcus MGAS8232 [26 32 23 18] [24 37 3025] [25 31 29 31] pyogenes Streptococcus MGAS315 [26 32 23 18] [24 37 3025] [25 31 29 31] pyogenes Streptococcus SSI-1 [26 32 23 18] [24 37 3025] [25 31 29 31] pyogenes Streptococcus MGAS10394 [26 32 23 18] [24 3730 25] [25 31 29 31] pyogenes Streptococcus Manfredo (M5) [26 32 23 18][24 37 30 25] [25 31 29 31] pyogenes Streptococcus SF370 (M1) [26 32 2318] [24 37 30 25] [25 31 29 31] pyogenes Streptococcus 670 [26 32 23 18][25 35 28 28] [25 32 29 30] pneumoniae Streptococcus R6 [26 32 23 18][25 35 28 28] [25 32 29 30] pneumoniae Streptococcus TIGR4 [26 32 23 18][25 35 28 28] [25 32 30 29] pneumoniae Streptococcus NCTC7868 [25 33 2318] [24 36 31 25] [25 31 29 31] gordonii Streptococcus NCTC 12261 [26 3223 18] [25 35 30 26] [25 32 29 30] mitis [24 31 35 29]* StreptococcusUA159 [24 32 24 19] [25 37 30 24] [28 31 26 31] mutans

TABLE 7B Base Compositions of Common Respiratory Pathogens for BioagentIdentifying Amplicons Corresponding to Primer Pair Nos: 349, 360, and356 Primer 349 Primer 360 Primer 356 Organism Strain [A G C T] [A G C T][A G C T] Klebsiella MGH78578 [25 31 25 22] [33 37 25 27] NO DATApneumoniae Yersinia pestis CO-92 Biovar [25 31 27 20] [34 35 25 28] NODATA Orientalis [25 32 26 20]* Yersinia pestis KIM5 P12 (Biovar [25 3127 20] [34 35 25 28] NO DATA Mediaevalis) [25 32 26 20]* Yersinia pestis91001 [25 31 27 20] [34 35 25 28] NO DATA Haemophilus KW20 [28 28 25 20][32 38 25 27] NO DATA influenzae Pseudomonas PAO1 [24 31 26 20] [31 3627 27] NO DATA aeruginosa [31 36 27 28]* Pseudomonas Pf0-1 NO DATA [3037 27 28] NO DATA fluorescens [30 37 27 28] Pseudomonas KT2440 [24 31 2620] [30 37 27 28] NO DATA putida Legionella Philadelphia-1 [23 30 25 23][30 39 29 24] NO DATA pneumophila Francisella schu 4 [26 31 25 19] [3236 27 27] NO DATA tularensis Bordetella Tohama I [21 29 24 18] [33 36 2627] NO DATA pertussis Burkholderia J2315 [23 27 22 20] [31 37 28 26] NODATA cepacia Burkholderia K96243 [23 27 22 20] [31 37 28 26] NO DATApseudomallei Neisseria FA 1090, ATCC 700825 [24 27 24 17] [34 37 25 26]NO DATA gonorrhoeae Neisseria MC58 (serogroup B) [25 27 22 18] [34 37 2526] NO DATA meningitidis Neisseria serogroup C, FAM18 [25 26 23 18] [3437 25 26] NO DATA meningitidis Neisseria Z2491 (serogroup A) [25 26 2318] [34 37 25 26] NO DATA meningitidis Chlamydophila TW-183 [30 28 2718] NO DATA NO DATA pneumoniae Chlamydophila AR39 [30 28 27 18] NO DATANO DATA pneumoniae Chlamydophila CWL029 [30 28 27 18] NO DATA NO DATApneumoniae Chlamydophila J138 [30 28 27 18] NO DATA NO DATA pneumoniaeCorynebacterium NCTC13129 NO DATA [29 40 28 25] NO DATA diphtheriaeMycobacterium k10 NO DATA [33 35 32 22] NO DATA avium Mycobacterium 104NO DATA [33 35 32 22] NO DATA avium Mycobacterium CSU#93 NO DATA [30 3634 22] NO DATA tuberculosis Mycobacterium CDC 1551 NO DATA [30 36 34 22]NO DATA tuberculosis Mycobacterium H37Rv (lab strain) NO DATA [30 36 3422] NO DATA tuberculosis Mycoplasma M129 [28 30 24 19] [34 31 29 28] NODATA pneumoniae Staphylococcus MRSA252 [26 30 25 20] [31 38 24 29] [3330 31 27] aureus Staphylococcus MSSA476 [26 30 25 20] [31 38 24 29] [3330 31 27] aureus Staphylococcus COL [26 30 25 20] [31 38 24 29] [33 3031 27] aureus Staphylococcus Mu50 [26 30 25 20] [31 38 24 29] [33 30 3127] aureus Staphylococcus MW2 [26 30 25 20] [31 38 24 29] [33 30 31 27]aureus Staphylococcus N315 [26 30 25 20] [31 38 24 29] [33 30 31 27]aureus Staphylococcus NCTC 8325 [26 30 25 20] [31 38 24 29] [33 30 3127] aureus Streptococcus NEM316 [28 31 22 20] [33 37 24 28] [37 30 2826] agalactiae Streptococcus NC_002955 [28 31 23 19] [33 38 24 27] [3731 28 25] equi Streptococcus MGAS8232 [28 31 23 19] [33 37 24 28] [38 3129 23] pyogenes Streptococcus MGAS315 [28 31 23 19] [33 37 24 28] [38 3129 23] pyogenes Streptococcus SSI-1 [28 31 23 19] [33 37 24 28] [38 3129 23] pyogenes Streptococcus MGAS10394 [28 31 23 19] [33 37 24 28] [3831 29 23] pyogenes Streptococcus Manfredo (M5) [28 31 23 19] [33 37 2428] [38 31 29 23] pyogenes Streptococcus SF370 (M1) [28 31 23 19] [33 3724 28] [38 31 29 23] pyogenes [28 31 22 20]* Streptococcus 670 [28 31 2220] [34 36 24 28] [37 30 29 25] pneumoniae Streptococcus R6 [28 31 2220] [34 36 24 28] [37 30 29 25] pneumoniae Streptococcus TIGR4 [28 31 2220] [34 36 24 28] [37 30 29 25] pneumoniae Streptococcus NCTC7868 [28 3223 20] [34 36 24 28] [36 31 29 25] gordonii Streptococcus NCTC 12261 [2831 22 20] [34 36 24 28] [37 30 29 25] mitis [29 30 22 20]* StreptococcusUA159 [26 32 23 22] [34 37 24 27] NO DATA mutans

TABLE 7C Base Compositions of Common Respiratory Pathogens for BioagentIdentifying Amplicons Corresponding to Primer Pair Nos: 449, 354, and352 Primer 449 Primer 354 Primer 352 Organism Strain [A G C T] [A G C T][A G C T] Klebsiella MGH78578 NO DATA [27 33 36 26] NO DATA pneumoniaeYersinia pestis CO-92 Biovar NO DATA [29 31 33 29] [32 28 20 25]Orientalis Yersinia pestis KIM5 P12 (Biovar NO DATA [29 31 33 29] [32 2820 25] Mediaevalis) Yersinia pestis 91001 NO DATA [29 31 33 29] NO DATAHaemophilus KW20 NO DATA [30 29 31 32] NO DATA influenzae PseudomonasPAO1 NO DATA [26 33 39 24] NO DATA aeruginosa Pseudomonas Pf0-1 NO DATA[26 33 34 29] NO DATA fluorescens Pseudomonas KT2440 NO DATA [25 34 3627] NO DATA putida Legionella Philadelphia-1 NO DATA NO DATA NO DATApneumophila Francisella schu 4 NO DATA [33 32 25 32] NO DATA tularensisBordetella Tohama I NO DATA [26 33 39 24] NO DATA pertussis BurkholderiaJ2315 NO DATA [25 37 33 27] NO DATA cepacia Burkholderia K96243 NO DATA[25 37 34 26] NO DATA pseudomallei Neisseria FA 1090, ATCC 700825 [17 2322 10] [29 31 32 30] NO DATA gonorrhoeae Neisseria MC58 (serogroup B) NODATA [29 30 32 31] NO DATA meningitidis Neisseria serogroup C, FAM18 NODATA [29 30 32 31] NO DATA meningitidis Neisseria Z2491 (serogroup A) NODATA [29 30 32 31] NO DATA meningitidis Chlamydophila TW-183 NO DATA NODATA NO DATA pneumoniae Chlamydophila AR39 NO DATA NO DATA NO DATApneumoniae Chlamydophila CWL029 NO DATA NO DATA NO DATA pneumoniaeChlamydophila J138 NO DATA NO DATA NO DATA pneumoniae CorynebacteriumNCTC13129 NO DATA NO DATA NO DATA diphtheriae Mycobacterium k10 NO DATANO DATA NO DATA avium Mycobacterium 104 NO DATA NO DATA NO DATA aviumMycobacterium CSU#93 NO DATA NO DATA NO DATA tuberculosis MycobacteriumCDC 1551 NO DATA NO DATA NO DATA tuberculosis Mycobacterium H37Rv (labstrain) NO DATA NO DATA NO DATA tuberculosis Mycoplasma M129 NO DATA NODATA NO DATA pneumoniae Staphylococcus MRSA252 [17 20 21 17] [30 27 3035] [36 24 19 26] aureus Staphylococcus MSSA476 [17 20 21 17] [30 27 3035] [36 24 19 26] aureus Staphylococcus COL [17 20 21 17] [30 27 30 35][35 24 19 27] aureus Staphylococcus Mu50 [17 20 21 17] [30 27 30 35] [3624 19 26] aureus Staphylococcus MW2 [17 20 21 17] [30 27 30 35] [36 2419 26] aureus Staphylococcus N315 [17 20 21 17] [30 27 30 35] [36 24 1926] aureus Staphylococcus NCTC 8325 [17 20 21 17] [30 27 30 35] [35 2419 27] aureus Streptococcus NEM316 [22 20 19 14] [26 31 27 38] [29 26 2228] agalactiae Streptococcus NC_002955 [22 21 19 13] NO DATA NO DATAequi Streptococcus MGAS8232 [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus MGAS315 [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus SSI-1 [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus MGAS10394 [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus Manfredo (M5) [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus SF370 (M1) [23 21 19 12] [24 32 30 36] NO DATA pyogenesStreptococcus 670 [22 20 19 14] [25 33 29 35] [30 29 21 25] pneumoniaeStreptococcus R6 [22 20 19 14] [25 33 29 35] [30 29 21 25] pneumoniaeStreptococcus TIGR4 [22 20 19 14] [25 33 29 35] [30 29 21 25] pneumoniaeStreptococcus NCTC7868 [21 21 19 14] NO DATA [29 26 22 28] gordoniiStreptococcus NCTC 12261 [22 20 19 14] [26 30 32 34] NO DATA mitisStreptococcus UA159 NO DATA NO DATA NO DATA mutans

TABLE 7D Base Compositions of Common Respiratory Pathogens for BioagentIdentifying Amplicons Corresponding to Primer Pair Nos: 355, 358, and359 Primer 355 Primer 358 Primer 359 Organism Strain [A G C T] [A G C T][A G C T] Klebsiella MGH78578 NO DATA [24 39 33 20] [25 21 24 17]pneumoniae Yersinia pestis CO-92 Biovar NO DATA [26 34 35 21] [23 23 1922] Orientalis Yersinia pestis KIM5 P12 (Biovar NO DATA [26 34 35 21][23 23 19 22] Mediaevalis) Yersinia pestis 91001 NO DATA [26 34 35 21][23 23 19 22] Haemophilus KW20 NO DATA NO DATA NO DATA influenzaePseudomonas PAO1 NO DATA NO DATA NO DATA aeruginosa Pseudomonas Pf0-1 NODATA NO DATA NO DATA fluorescens Pseudomonas KT2440 NO DATA [21 37 3721] NO DATA putida Legionella Philadelphia-1 NO DATA NO DATA NO DATApneumophila Francisella schu 4 NO DATA NO DATA NO DATA tularensisBordetella Tohama I NO DATA NO DATA NO DATA pertussis Burkholderia J2315NO DATA NO DATA NO DATA cepacia Burkholderia K96243 NO DATA NO DATA NODATA pseudomallei Neisseria FA 1090, ATCC 700825 NO DATA NO DATA NO DATAgonorrhoeae Neisseria MC58 (serogroup B) NO DATA NO DATA NO DATAmeningitidis Neisseria serogroup C, FAM18 NO DATA NO DATA NO DATAmeningitidis Neisseria Z2491 (serogroup A) NO DATA NO DATA NO DATAmeningitidis Chlamydophila TW-183 NO DATA NO DATA NO DATA pneumoniaeChlamydophila AR39 NO DATA NO DATA NO DATA pneumoniae ChlamydophilaCWL029 NO DATA NO DATA NO DATA pneumoniae Chlamydophila J138 NO DATA NODATA NO DATA pneumoniae Corynebacterium NCTC13129 NO DATA NO DATA NODATA diphtheriae Mycobacterium k10 NO DATA NO DATA NO DATA aviumMycobacterium 104 NO DATA NO DATA NO DATA avium Mycobacterium CSU#93 NODATA NO DATA NO DATA tuberculosis Mycobacterium CDC 1551 NO DATA NO DATANO DATA tuberculosis Mycobacterium H37Rv (lab strain) NO DATA NO DATA NODATA tuberculosis Mycoplasma M129 NO DATA NO DATA NO DATA pneumoniaeStaphylococcus MRSA252 NO DATA NO DATA NO DATA aureus StaphylococcusMSSA476 NO DATA NO DATA NO DATA aureus Staphylococcus COL NO DATA NODATA NO DATA aureus Staphylococcus Mu50 NO DATA NO DATA NO DATA aureusStaphylococcus MW2 NO DATA NO DATA NO DATA aureus Staphylococcus N315 NODATA NO DATA NO DATA aureus Staphylococcus NCTC 8325 NO DATA NO DATA NODATA aureus Streptococcus NEM316 NO DATA NO DATA NO DATA agalactiaeStreptococcus NC_002955 NO DATA NO DATA NO DATA equi StreptococcusMGAS8232 NO DATA NO DATA NO DATA pyogenes Streptococcus MGAS315 NO DATANO DATA NO DATA pyogenes Streptococcus SSI-1 NO DATA NO DATA NO DATApyogenes Streptococcus MGAS10394 NO DATA NO DATA NO DATA pyogenesStreptococcus Manfredo (M5) NO DATA NO DATA NO DATA pyogenesStreptococcus SF370 (M1) NO DATA NO DATA NO DATA pyogenes Streptococcus670 NO DATA NO DATA NO DATA pneumoniae Streptococcus R6 NO DATA NO DATANO DATA pneumoniae Streptococcus TIGR4 NO DATA NO DATA NO DATApneumoniae Streptococcus NCTC7868 NO DATA NO DATA NO DATA gordoniiStreptococcus NCTC 12261 NO DATA NO DATA NO DATA mitis StreptococcusUA159 NO DATA NO DATA NO DATA mutans

TABLE 7E Base Compositions of Common Respiratory Pathogens for BioagentIdentifying Amplicons Corresponding to Primer Pair Nos: 362, 363, and367 Primer 362 Primer 363 Primer 367 Organism Strain [A G C T] [A G C T][A G C T] Klebsiella MGH78578 [21 33 22 16] [16 34 26 26] NO DATApneumoniae Yersinia pestis CO-92 Biovar [20 34 18 20] NO DATA NO DATAOrientalis Yersinia pestis KIM5 P12 (Biovar [20 34 18 20] NO DATA NODATA Mediaevalis) Yersinia pestis 91001 [20 34 18 20] NO DATA NO DATAHaemophilus KW20 NO DATA NO DATA NO DATA influenzae Pseudomonas PAO1 [1935 21 17] [16 36 28 22] NO DATA aeruginosa Pseudomonas Pf0-1 NO DATA [1835 26 23] NO DATA fluorescens Pseudomonas KT2440 NO DATA [16 35 28 23]NO DATA putida Legionella Philadelphia-1 NO DATA NO DATA NO DATApneumophila Francisella schu 4 NO DATA NO DATA NO DATA tularensisBordetella Tohama I [20 31 24 17] [15 34 32 21] [26 25 34 19] pertussisBurkholderia J2315 [20 33 21 18] [15 36 26 25] [25 27 32 20] cepaciaBurkholderia K96243 [19 34 19 20] [15 37 28 22] [25 27 32 20]pseudomallei Neisseria FA 1090, ATCC 700825 NO DATA NO DATA NO DATAgonorrhoeae Neisseria MC58 (serogroup B) NO DATA NO DATA NO DATAmeningitidis Neisseria serogroup C, FAM18 NO DATA NO DATA NO DATAmeningitidis Neisseria Z2491 (serogroup A) NO DATA NO DATA NO DATAmeningitidis Chlamydophila TW-183 NO DATA NO DATA NO DATA pneumoniaeChlamydophila AR39 NO DATA NO DATA NO DATA pneumoniae ChlamydophilaCWL029 NO DATA NO DATA NO DATA pneumoniae Chlamydophila J138 NO DATA NODATA NO DATA pneumoniae Corynebacterium NCTC13129 NO DATA NO DATA NODATA diphtheriae Mycobacterium k10 [19 34 23 16] NO DATA [24 26 35 19]avium Mycobacterium 104 [19 34 23 16] NO DATA [24 26 35 19] aviumMycobacterium CSU#93 [19 31 25 17] NO DATA [25 25 34 20] tuberculosisMycobacterium CDC 1551 [19 31 24 18] NO DATA [25 25 34 20] tuberculosisMycobacterium H37Rv (lab strain) [19 31 24 18] NO DATA [25 25 34 20]tuberculosis Mycoplasma M129 NO DATA NO DATA NO DATA pneumoniaeStaphylococcus MRSA252 NO DATA NO DATA NO DATA aureus StaphylococcusMSSA476 NO DATA NO DATA NO DATA aureus Staphylococcus COL NO DATA NODATA NO DATA aureus Staphylococcus Mu50 NO DATA NO DATA NO DATA aureusStaphylococcus MW2 NO DATA NO DATA NO DATA aureus Staphylococcus N315 NODATA NO DATA NO DATA aureus Staphylococcus NCTC 8325 NO DATA NO DATA NODATA aureus Streptococcus NEM316 NO DATA NO DATA NO DATA agalactiaeStreptococcus NC_002955 NO DATA NO DATA NO DATA equi StreptococcusMGAS8232 NO DATA NO DATA NO DATA pyogenes Streptococcus MGAS315 NO DATANO DATA NO DATA pyogenes Streptococcus SSI-1 NO DATA NO DATA NO DATApyogenes Streptococcus MGAS10394 NO DATA NO DATA NO DATA pyogenesStreptococcus Manfredo (M5) NO DATA NO DATA NO DATA pyogenesStreptococcus SF370 (M1) NO DATA NO DATA NO DATA pyogenes Streptococcus670 NO DATA NO DATA NO DATA pneumoniae Streptococcus R6 [20 30 19 23] NODATA NO DATA pneumoniae Streptococcus TIGR4 [20 30 19 23] NO DATA NODATA pneumoniae Streptococcus NCTC7868 NO DATA NO DATA NO DATA gordoniiStreptococcus NCTC 12261 NO DATA NO DATA NO DATA mitis StreptococcusUA159 NO DATA NO DATA NO DATA mutans

Four sets of throat samples from military recruits at different militaryfacilities taken at different time points were analyzed using theprimers of the present invention. The first set was collected at amilitary training center from November 1 to Dec. 20, 2002 during one ofthe most severe outbreaks of pneumonia associated with group AStreptococcus in the United States since 1968. During this outbreak,fifty-one throat swabs were taken from both healthy and hospitalizedrecruits and plated on blood agar for selection of putative group AStreptococcus colonies. A second set of 15 original patient specimenswas taken during the height of this group A Streptococcus-associatedrespiratory disease outbreak. The third set were historical samples,including twenty-seven isolates of group A Streptococcus, from diseaseoutbreaks at this and other military training facilities during previousyears. The fourth set of samples was collected from five geographicallyseparated military facilities in the continental U.S. in the winterimmediately following the severe November/December 2002 outbreak.

Pure colonies isolated from group A Streptococcus-selective media fromall four collection periods were analyzed with the surveillance primerset. All samples showed base compositions that precisely matched thefour completely sequenced strains of Streptococcus pyogenes. Shown inFIG. 4 is a 3D diagram of base composition (axes A, G and C) of bioagentidentifying amplicons obtained with primer pair number 14 (a precursorof primer pair number 348 which targets 16S rRNA). The diagram indicatesthat the experimentally determined base compositions of the clinicalsamples closely match the base compositions expected for Streptococcuspyogenes and are distinct from the expected base compositions of otherorganisms.

In addition to the identification of Streptococcus pyogenes, otherpotentially pathogenic organisms were identified concurrently. Massspectral analysis of a sample whose nucleic acid was amplified by primerpair number 349 (SEQ ID NOs: 401:1156) exhibited signals of bioagentidentifying amplicons with molecular masses that were found tocorrespond to analogous base compositions of bioagent identifyingamplicons of Streptococcus pyogenes (A27 G32 C24 T18), Neisseriameningitidis (A25 G27 C22 T18), and Haemophilus influenzae (A28 G28 C25T20) (see FIG. 5 and Table 7B). These organisms were present in a ratioof 4:5:20 as determined by comparison of peak heights with peak heightof an internal PCR calibration standard as described in commonly ownedU.S. Patent Application Ser. No. 60/545,425 which is incorporated hereinby reference in its entirety.

Since certain division-wide primers that target housekeeping genes aredesigned to provide coverage of specific divisions of bacteria toincrease the confidence level for identification of bacterial species,they are not expected to yield bioagent identifying amplicons fororganisms outside of the specific divisions. For example, primer pairnumber 356 (SEQ ID NOs: 449:1380) primarily amplifies the nucleic acidof members of the classes Bacilli and Clostridia and is not expected toamplify proteobacteria such as Neisseria meningitidis and Haemophilusinfluenzae. As expected, analysis of the mass spectrum of amplificationproducts obtained with primer pair number 356 does not indicate thepresence of Neisseria meningitidis and Haemophilus influenzae but doesindicate the presence of Streptococcus pyogenes (FIGS. 3 and 6, Table7B). Thus, these primers or types of primers can confirm the absence ofparticular bioagents from a sample.

The 15 throat swabs from military recruits were found to contain arelatively small set of microbes in high abundance. The most common wereHaemophilus influenza, Neisseria meningitides, and Streptococcuspyogenes. Staphylococcus epidermidis, Moraxella cattarhalis,Corynebacterium pseudodiphtheriticum, and Staphylococcus aureus werepresent in fewer samples. An equal number of samples from healthyvolunteers from three different geographic locations, were identicallyanalyzed. Results indicated that the healthy volunteers have bacterialflora dominated by multiple, commensal non-beta-hemolytic Streptococcalspecies, including the viridans group streptococci (S. parasangunis, S.vestibularis, S. mitis, S. oralis and S. pneumoniae; data not shown),and none of the organisms found in the military recruits were found inthe healthy controls at concentrations detectable by mass spectrometry.Thus, the military recruits in the midst of a respiratory diseaseoutbreak had a dramatically different microbial population than thatexperienced by the general population in the absence of epidemicdisease.

Example 7 Triangulation Genotyping Analysis for Determination ofemm-Type of Streptococcus pyogenes in Epidemic Surveillance

As a continuation of the epidemic surveillance investigation of Example6, determination of sub-species characteristics (genotyping) ofStreptococcus pyogenes, was carried out based on a strategy thatgenerates strain-specific signatures according to the rationale ofMulti-Locus Sequence Typing (MLST). In classic MLST analysis, internalfragments of several housekeeping genes are amplified and sequenced(Enright et al. Infection and Immunity, 2001, 69, 2416-2427). In classicMLST analysis, internal fragments of several housekeeping genes areamplified and sequenced. In the present investigation, bioagentidentifying amplicons from housekeeping genes were produced usingdrill-down primers and analyzed by mass spectrometry. Since massspectral analysis results in molecular mass, from which base compositioncan be determined, the challenge was to determine whether resolution ofemm classification of strains of Streptococcus pyogenes could bedetermined.

For the purpose of development of a triangulation genotyping assay, analignment was constructed of concatenated alleles of seven MLSThousekeeping genes (glucose kinase (gki), glutamine transporter protein(gtr), glutamate racemase (murI), DNA mismatch repair protein (mutS),xanthine phosphoribosyl transferase (xpt), and acetyl-CoA acetyltransferase (yqiL)) from each of the 212 previously emm-typed strains ofStreptococcus pyogenes. From this alignment, the number and location ofprimer pairs that would maximize strain identification via basecomposition was determined. As a result, 6 primer pairs were chosen asstandard drill-down primers for determination of emm-type ofStreptococcus pyogenes. These six primer pairs are displayed in Table 8.This drill-down set comprises primers with T modifications (note TMODdesignation in primer names) which constitutes a functional improvementwith regard to prevention of non-templated adenylation (vide supra)relative to originally selected primers which are displayed below in thesame row. TABLE 8 Triangulation Genotyping Analysis Primer Pairs forGroup A Streptococcus Drill-Down Forward Primer Primer (SEQ ID ReversePrimer Target Pair No. Forward Primer Name NO:) Reverse Primer Name (SEQID NO:) Gene 442 SP101_SPET11_358_387_TMOD_F 588SP101_SPET11_448_473_TMOD_R 998 gki 80 SP101_SPET11_358_387_F 126SP101_SPET11_448_473_TMOD_R 766 gki 443 SP101_SPET11_600_629_TMOD_F 348SP101_SPET11_686_714_TMOD_R 1018 gtr 81 SP101_SPET11_600_629_F 62SP101_SPET11_686_714_R 772 gtr 426 SP101_SPET11_1314_1336_TMOD_F 363SP101_SPET11_1403_1431_TMOD_R 849 murI 86 SP101_SPET11_1314_1336_F 68SP101_SPET11_1403_1431_R 711 murI 430 SP101_SPET11_1807_1835_TMOD_F 235SP101_SPET11_1901_1927_TMOD_R 1439 mutS 90 SP101_SPET11_1807_1835_F 33SP101_SPET11_1901_1927_R 1412 mutS 438 SP101_SPET11_3075_3103_TMOD_F 473SP101_SPET11_3168_3196_TMOD_R 875 xpt 96 SP101_SPET11_3075_3103_F 108SP101_SPET11_3168_3196_R 715 xpt 441 SP101_SPET11_3511_3535_TMOD_F 531SP101_SPET11_3605_3629_TMOD_R 1294 yqiL 98 SP101_SPET11_3511_3535_F 116SP101_SPET11_3605_3629_R 832 yqiL

The primers of Table 8 were used to produce bioagent identifyingamplicons from nucleic acid present in the clinical samples. Thebioagent identifying amplicons which were subsequently analyzed by massspectrometry and base compositions corresponding to the molecular masseswere calculated.

Of the 51 samples taken during the peak of the November/December 2002epidemic (Table 9A-C rows 1-3), all except three samples were found torepresent emm3, a Group A Streptococcus genotype previously associatedwith high respiratory virulence. The three outliers were from samplesobtained from healthy individuals and probably represent non-epidemicstrains. Archived samples (Tables 9A-C rows 5-13) from historicalcollections showed a greater heterogeneity of base compositions and emmtypes as would be expected from different epidemics occurring atdifferent places and dates. The results of the mass spectrometryanalysis and emm gene sequencing were found to be concordant for theepidemic and historical samples. TABLE 9A Base Composition Analysis ofBioagent Identifying Amplicons of Group A Streptococcus samples from SixMilitary Installations Obtained with Primer Pair Nos. 426 and 430emm-type by murI mutS # of Mass emm-Gene Location (Primer Pair (PrimerPair Instances Spectrometry Sequencing (sample) Year No. 426) No. 430)48  3 3 MCRD San 2002 A39 G25 C20 T34 A38 G27 C23 T33 2 6 6 Diego A40G24 C20 T34 A38 G27 C23 T33 1 28  28  (Cultured) A39 G25 C20 T34 A38 G27C23 T33 15  3 ND A39 G25 C20 T34 A38 G27 C23 T33 6 3 3 NHRC San 2003 A39G25 C20 T34 A38 G27 C23 T33 3 5, 58 5 Diego- A40 G24 C20 T34 A38 G27 C23T33 6 6 6 Archive A40 G24 C20 T34 A38 G27 C23 T33 1 11  11  (Cultured)A39 G25 C20 T34 A38 G27 C23 T33 3 12  12  A40 G24 C20 T34 A38 G26 C24T33 1 22  22  A39 G25 C20 T34 A38 G27 C23 T33 3 25, 75  75  A39 G25 C20T34 A38 G27 C23 T33 4 44/61, 82, 9 44/61 A40 G24 C20 T34 A38 G26 C24 T332 53, 91 91 A39 G25 C20 T34 A38 G27 C23 T33 1 2 2 Ft. 2003 A39 G25 C20T34 A38 G27 C24 T32 2 3 3 Leonard A39 G25 C20 T34 A38 G27 C23 T33 1 4 4Wood A39 G25 C20 T34 A38 G27 C23 T33 1 6 6 (Cultured) A40 G24 C20 T34A38 G27 C23 T33 11  25 or 75 75  A39 G25 C20 T34 A38 G27 C23 T33 1 25,75, 33, 75  A39 G25 C20 T34 A38 G27 C23 T33 34, 4, 52, 84 1 44/61 or 8244/61 A40 G24 C20 T34 A38 G26 C24 T33 or 9 2 5 or 58 5 A40 G24 C20 T34A38 G27 C23 T33 3 1 1 Ft. Sill 2003 A40 G24 C20 T34 A38 G27 C23 T33 2 33 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 1 4 4 A39 G25 C20 T34 A38G27 C23 T33 1 28  28  A39 G25 C20 T34 A38 G27 C23 T33 1 3 3 Ft. 2003 A39G25 C20 T34 A38 G27 C23 T33 1 4 4 Benning A39 G25 C20 T34 A38 G27 C23T33 3 6 6 (Cultured) A40 G24 C20 T34 A38 G27 C23 T33 1 11  11  A39 G25C20 T34 A38 G27 C23 T33 1 13   94** A40 G24 C20 T34 A38 G27 C23 T33 144/61 or 82 82  A40 G24 C20 T34 A38 G26 C24 T33 or 9 1 5 or 58  58  A40G24 C20 T34 A38 G27 C23 T33 1 78 or 89  89  A39 G25 C20 T34 A38 G27 C23T33 2 5 or 58 ND Lackland 2003 A40 G24 C20 T34 A38 G27 C23 T33 1 2 AFBA39 G25 C20 T34 A38 G27 C24 T32 1 81 or 90 (Throat A40 G24 C20 T34 A38G27 C23 T33 1 78  Swabs) A38 G26 C20 T34 A38 G27 C23 T33   3*** Nodetection No detection No detection 7 3 ND MCRD San 2002 A39 G25 C20 T34A38 G27 C23 T33 1 3 ND Diego No detection A38 G27 C23 T33 1 3 ND (ThroatNo detection No detection 1 3 ND Swabs) No detection No detection 2 3 NDNo detection A38 G27 C23 T33 3 No detection ND No detection No detection

TABLE 9B Base Composition Analysis of Bioagent Identifying Amplicons ofGroup A Streptococcus samples from Six Military Installations Obtainedwith Primer Pair Nos. 438 and 441 emm-type by xpt yqiL # of Massemm-Gene Location (Primer Pair (Primer Pair Instances SpectrometrySequencing (sample) Year No. 438) No. 441) 48  3 3 MCRD San 2002 A30 G36C20 T36 A40 G29 C19 T31 2 6 6 Diego A30 G36 C20 T36 A40 G29 C19 T31 128  28  (Cultured) A30 G36 C20 T36 A41 G28 C18 T32 15  3 ND A30 G36 C20T36 A40 G29 C19 T31 6 3 3 NHRC San 2003 A30 G36 C20 T36 A40 G29 C19 T313 5, 58 5 Diego- A30 G36 C20 T36 A40 G29 C19 T31 6 6 6 Archive A30 G36C20 T36 A40 G29 C19 T31 1 11  11  (Cultured) A30 G36 C20 T36 A40 G29 C19T31 3 12  12  A30 G36 C19 T37 A40 G29 C19 T31 1 22  22  A30 G36 C20 T36A40 G29 C19 T31 3 25, 75 75  A30 G36 C20 T36 A40 G29 C19 T31 4 44/61,82, 9 44/61 A30 G36 C20 T36 A41 G28 C19 T31 2 53, 91 91  A30 G36 C19 T37A40 G29 C19 T31 1 2 2 Ft. 2003 A30 G36 C20 T36 A40 G29 C19 T31 2 3 3Leonard A30 G36 C20 T36 A40 G29 C19 T31 1 4 4 Wood A30 G36 C19 T37 A41G28 C19 T31 1 6 6 (Cultured) A30 G36 C20 T36 A40 G29 C19 T31 11  25 or75 75  A30 G36 C20 T36 A40 G29 C19 T31 1 25, 75, 33, 75  A30 G36 C19 T37A40 G29 C19 T31 34, 4, 52, 84 1 44/61 or 82 44/61 A30 G36 C20 T36 A41G28 C19 T31 or 9 2 5 or 58 5 A30 G36 C20 T36 A40 G29 C19 T31 3 1 1 Ft.Sill 2003 A30 G36 C19 T37 A40 G29 C19 T31 2 3 3 (Cultured) A30 G36 C20T36 A40 G29 C19 T31 1 4 4 A30 G36 C19 T37 A41 G28 C19 T31 1 28  28  A30G36 C20 T36 A41 G28 C18 T32 1 3 3 Ft. 2003 A30 G36 C20 T36 A40 G29 C19T31 1 4 4 Benning A30 G36 C19 T37 A41 G28 C19 T31 3 6 6 (Cultured) A30G36 C20 T36 A40 G29 C19 T31 1 11  11  A30 G36 C20 T36 A40 G29 C19 T31 113   94** A30 G36 C20 T36 A41 G28 C19 T31 1 44/61 or 82 82  A30 G36 C20T36 A41 G28 C19 T31 or 9 1 5 or 58 58  A30 G36 C20 T36 A40 G29 C19 T31 178 or 89 89  A30 G36 C20 T36 A41 G28 C19 T31 2 5 or 58 ND Lackland 2003A30 G36 C20 T36 A40 G29 C19 T31 1 2 AFB A30 G36 C20 T36 A40 G29 C19 T311 81 or 90 (Throat A30 G36 C20 T36 A40 G29 C19 T31 1 78  Swabs) A30 G36C20 T36 A41 G28 C19 T31   3*** No detection No detection No detection 73 ND MCRD San 2002 A30 G36 C20 T36 A40 G29 C19 T31 1 3 ND Diego A30 G36C20 T36 A40 G29 C19 T31 1 3 ND (Throat A30 G36 C20 T36 No detection 1 3ND Swabs) No detection A40 G29 C19 T31 2 3 ND A30 G36 C20 T36 A40 G29C19 T31 3 No detection ND No detection No detection

TABLE 9C Base Composition Analysis of Bioagent Identifying Amplicons ofGroup A Streptococcus samples from Six Military Installations Obtainedwith Primer Pair Nos. 438 and 441 emm-type by gki gtr # of Mass emm-GeneLocation (Primer Pair ((Primer Pair Instances Spectrometry Sequencing(sample) Year No. 442) No. 443) 48  3 3 MCRD San 2002 A32 G35 C17 T32A39 G28 C16 T32 2 6 6 Diego A31 G35 C17 T33 A39 G28 C15 T33 1 28  28 (Cultured) A30 G36 C17 T33 A39 G28 C16 T32 15  3 ND A32 G35 C17 T32 A39G28 C16 T32 6 3 3 NHRC San 2003 A32 G35 C17 T32 A39 G28 C16 T32 3 5, 585 Diego- A30 G36 C20 T30 A39 G28 C15 T33 6 6 6 Archive A31 G35 C17 T33A39 G28 C15 T33 1 11  11  (Cultured) A30 G36 C20 T30 A39 G28 C16 T32 312  12  A31 G35 C17 T33 A39 G28 C15 T33 1 22  22  A31 G35 C17 T33 A38G29 C15 T33 3 25, 75 75  A30 G36 C17 T33 A39 G28 C15 T33 4 44/61, 82, 944/61 A30 G36 C18 T32 A39 G28 C15 T33 2 53, 91 91  A32 G35 C17 T32 A39G28 C16 T32 1 2 2 Ft. 2003 A30 G36 C17 T33 A39 G28 C15 T33 2 3 3 LeonardA32 G35 C17 T32 A39 G28 C16 T32 1 4 4 Wood A31 G35 C17 T33 A39 G28 C15T33 1 6 6 (Cultured) A31 G35 C17 T33 A39 G28 C15 T33 11  25 or 75 75 A30 G36 C17 T33 A39 G28 C15 T33 1 25, 75, 33, 75  A30 G36 C17 T33 A39G28 C15 T33 34, 4, 52, 84 1 44/61 or 82 44/61 A30 G36 C18 T32 A39 G28C15 T33 or 9 2 5 or 58 5 A30 G36 C20 T30 A39 G28 C15 T33 3 1 1 Ft. Sill2003 A30 G36 C18 T32 A39 G28 C15 T33 2 3 3 (Cultured) A32 G35 C17 T32A39 G28 C16 T32 1 4 4 A31 G35 C17 T33 A39 G28 C15 T33 1 28  28  A30 G36C17 T33 A39 G28 C16 T32 1 3 3 Ft. 2003 A32 G35 C17 T32 A39 G28 C16 T32 14 4 Benning A31 G35 C17 T33 A39 G28 C15 T33 3 6 6 (Cultured) A31 G35 C17T33 A39 G28 C15 T33 1 11  11  A30 G36 C20 T30 A39 G28 C16 T32 1 13  94** A30 G36 C19 T31 A39 G28 C15 T33 1 44/61 or 82 82  A30 G36 C18 T32A39 G28 C15 T33 or 9 1 5 or 58 58  A30 G36 C20 T30 A39 G28 C15 T33 1 78or 89 89  A30 G36 C18 T32 A39 G28 C15 T33 2 5 or 58 ND Lackland 2003 A30G36 C20 T30 A39 G28 C15 T33 1 2 AFB A30 G36 C17 T33 A39 G28 C15 T33 1 81or 90 (Throat A30 G36 C17 T33 A39 G28 C15 T33 1 78  Swabs) A30 G36 C18T32 A39 G28 C15 T33   3*** No detection No detection No detection 7 3 NDMCRD San 2002 A32 G35 C17 T32 A39 G28 C16 T32 1 3 ND Diego No detectionNo detection 1 3 ND (Throat A32 G35 C17 T32 A39 G28 C16 T32 1 3 NDSwabs) A32 G35 C17 T32 No detection 2 3 ND A32 G35 C17 T32 No detection3 No detection ND No detection No detection

Example 8 Design of Calibrant Polynucleotides Based on BioagentIdentifying Amplicons for Identification of Species of Bacteria(Bacterial Bioagent Identifying Amplicons)

This example describes the design of 19 calibrant polynucleotides basedon bacterial bioagent identifying amplicons corresponding to the primersof the broad surveillance set (Table 5) and the Bacillus anthracisdrill-down set (Table 6).

Calibration sequences were designed to simulate bacterial bioagentidentifying amplicons produced by the T modified primer pairs shown inTables 5 and 6 (primer names have the designation “TMOD”). Thecalibration sequences were chosen as a representative member of thesection of bacterial genome from specific bacterial species which wouldbe amplified by a given primer pair. The model bacterial species uponwhich the calibration sequences are based are also shown in Table 10.For example, the calibration sequence chosen to correspond to anamplicon produced by primer pair no. 361 is SEQ ID NO: 1445. In Table10, the forward (_F) or reverse (_R) primer name indicates thecoordinates of an extraction representing a gene of a standard referencebacterial genome to which the primer hybridizes e.g.: the forward primername 16S_EC_(—)713_(—)732_TMOD_F indicates that the forward primerhybridizes to residues 713-732 of the gene encoding 16S ribosomal RNA inan E. coli reference sequence (in this case, the reference sequence isan extraction consisting of residues 4033120-4034661 of the genomicsequence of E. coli K12 (GenBank gi number 16127994). Additional genecoordinate reference information is shown in Table 11. The designation“TMOD” in the primer names indicates that the 5′ end of the primer hasbeen modified with a non-matched template T residue which prevents thePCR polymerase from adding non-templated adenosine residues to the 5′end of the amplification product, an occurrence which may result inmiscalculation of base composition from molecular mass data (videsupra).

The 19 calibration sequences described in Tables 10 and 11 were combinedinto a single calibration polynucleotide sequence (SEQ ID NO: 1464—whichis herein designated a “combination calibration polynucleotide”) whichwas then cloned into a pCR®-Blunt vector (Invitrogen, Carlsbad, Calif.).This combination calibration polynucleotide can be used in conjunctionwith the primers of Tables 5 or 6 as an internal standard to producecalibration amplicons for use in determination of the quantity of anybacterial bioagent. Thus, for example, when the combination calibrationpolynucleotide vector is present in an amplification reaction mixture, acalibration amplicon based on primer pair 346 (16S rRNA) will beproduced in an amplification reaction with primer pair 346 and acalibration amplicon based on primer pair 363 (rpoC) will be producedwith primer pair 363. Coordinates of each of the 19 calibrationsequences within the calibration polynucleotide (SEQ ID NO: 1464) areindicated in Table 11. TABLE 10 Bacterial Primer Pairs for Production ofBacterial Bioagent Identifying Amplicons and CorrespondingRepresentative Calibration Sequences Forward Reverse Calibration PrimerPrimer Calibration Sequence Primer (SEQ ID (SEQ ID Sequence Model (SEQID Pair No. Forward Primer Name NO:) Reverse Primer Name NO:) SpeciesNO:) 361 16S_EC_1090_1111_2_TMOD_F 697 16S_EC_1175_1196_TMOD_R 1398Bacillus 1445 anthracis 346 16S_EC_713_732_TMOD_F 20216S_EC_789_809_TMOD_R 1110 Bacillus 1446 anthracis 34716S_EC_785_806_TMOD_F 560 16S_EC_880_897_TMOD_R 1278 Bacillus 1447anthracis 348 16S_EC_960_981_TMOD_F 706 16S_EC_1054_1073_TMOD_R 895Bacillus 1448 anthracis 349 23S_EC_1826_1843_TMOD_F 40123S_EC_1906_1924_TMOD_R 1156 Bacillus 1449 anthracis 36023S_EC_2646_2667_TMOD_F 409 23S_EC_2745_2765_TMOD_R 1434 Bacillus 1450anthracis 350 CAPC_BA_274_303_TMOD_F 476 CAPC_BA_349_376_TMOD_R 1314Bacillus 1451 anthracis 351 CYA_BA_1353_1379_TMOD_F 355CYA_BA_1448_1467_TMOD_R 1423 Bacillus 1452 anthracis 352INFB_EC_1365_1393_TMOD_F 687 INFB_EC_1439_1467_TMOD_R 1411 Bacillus 1453anthracis 353 LEF_BA_756_781_TMOD_F 220 LEF_BA_843_872_TMOD_R 1394Bacillus 1454 anthracis 356 RPLB_EC_650_679_TMOD_F 449RPLB_EC_739_762_TMOD_R 1380 Clostridium 1455 botulinum 449RPLB_EC_690_710_F 309 RPLB_EC_737_758_R 1336 Clostridium 1456 botulinum359 RPOB_EC_1845_1866_TMOD_F 659 RPOB_EC_1909_1929_TMOD_R 1250 Yersinia1457 Pestis 362 RPOB_EC_3799_3821_TMOD_F 581 RPOB_EC_3862_3888_TMOD_R1325 Burkholderia 1458 mallei 363 RPOC_EC_2146_2174_TMOD_F 284RPOC_EC_2227_2245_TMOD_R 898 Burkholderia 1459 mallei 354RPOC_EC_2218_2241_TMOD_F 405 RPOC_EC_2313_2337_TMOD_R 1072 Bacillus 1460anthracis 355 SSPE_BA_115_137_TMOD_F 255 SSPE_BA_197_222_TMOD_R 1402Bacillus 1461 anthracis 367 TUFB_EC_957_979_TMOD_F 308TUFB_EC_1034_1058_TMOD_R 1276 Burkholderia 1462 mallei 358VALS_EC_1105_1124_TMOD_F 385 VALS_EC_1195_1218_TMOD_R 1093 Yersinia 1463Pestis

TABLE 11 Primer Pair Gene Coordinate References and CalibrationPolynucleotide Sequence Coordinates within the Combination CalibrationPolynucleotide Coordinates of Gene Extraction Calibration Sequence inBacterial Coordinates Reference GenBank GI Combination Calibration Geneand of Genomic or Plasmid No. of Genomic (G) or Primer Polynucleotide(SEQ ID Species Sequence Plasmid (P) Sequence Pair No. NO: 1464) 16S E.coli 4033120 . . . 4034661 16127994 (G) 346  16 . . . 109 16S E. coli4033120 . . . 4034661 16127994 (G) 347  83 . . . 190 16S E. coli 4033120. . . 4034661 16127994 (G) 348 246 . . . 353 16S E. coli 4033120 . . .4034661 16127994 (G) 361 368 . . . 469 23S E. coli 4166220 . . . 416912316127994 (G) 349 743 . . . 837 23S E. coli 4166220 . . . 416912316127994 (G) 360 865 . . . 981 rpoB E. coli. 4178823 . . . 418285116127994 (G) 359 1591 . . . 1672 (complement strand) rpos E. coli.4178823 . . . 4182851 16127994 (G) 362 2081 . . . 2167 (complementstrand) rpoC E. coli 4182928 . . . 4187151 16127994 (G) 354 1810 . . .1926 rpoC E. coli 4182928 . . . 4187151 16127994 (G) 363 2183 . . . 2279infB E. coli. 3313655 . . . 3310983 16127994 (G) 352 1692 . . . 1791(complement strand) tufB E. coli 4173523 . . . 4174707 16127994 (G) 3672400 . . . 2498 rplB E. coli 3449001 . . . 3448180 16127994 (G) 356 1945. . . 2060 rplB E. coli 3449001 . . . 3448180 16127994 (G) 449 1986 . .. 2055 vals E. coli 4481405 . . . 4478550 16127994 (G) 358 1462 . . .1572 (complement strand) capC 56074 . . . 55628  6470151 (P) 350 2517 .. . 2616 B. anthracis (complement strand) cya 156626 . . . 154288 4894216 (P) 351 1338 . . . 1449 B. anthracis (complement strand) lef127442 . . . 129921  4894216 (P) 353 1121 . . . 1234 B. anthracis sspE226496 . . . 226783 30253828 (G) 355 1007-1104 B. anthracis

Example 9 Use of a Calibration Polynucleotide for Determining theQuantity of Bacillus Anthracis in a Sample Containing a Mixture ofMicrobes

The process described in this example is shown in FIG. 2. The capC geneis a gene involved in capsule synthesis which resides on the pX02plasmid of Bacillus anthracis. Primer pair number 350 (see Tables 10 and11) was designed to identify Bacillus anthracis via production of abacterial bioagent identifying amplicon. Known quantities of thecombination calibration polynucleotide vector described in Example 8were added to amplification mixtures containing bacterial bioagentnucleic acid from a mixture of microbes which included the Ames strainof Bacillus anthracis. Upon amplification of the bacterial bioagentnucleic acid and the combination calibration polynucleotide vector withprimer pair no. 350, bacterial bioagent identifying amplicons andcalibration amplicons were obtained and characterized by massspectrometry. A mass spectrum measured for the amplification reaction isshown in FIG. 7. The molecular masses of the bioagent identifyingamplicons provided the means for identification of the bioagent fromwhich they were obtained (Ames strain of Bacillus anthracis) and themolecular masses of the calibration amplicons provided the means fortheir identification as well. The relationship between the abundance(peak height) of the calibration amplicon signals and the bacterialbioagent identifying amplicon signals provides the means of calculationof the copies of the pX02 plasmid of the Ames strain of Bacillusanthracis. Methods of calculating quantities of molecules based oninternal calibration procedures are well known to those of ordinaryskill in the art.

Averaging the results of 10 repetitions of the experiment describedabove, enabled a calculation that indicated that the quantity of Amesstrain of Bacillus anthracis present in the sample corresponds toapproximately 10 copies of pX02 plasmid.

Example 10 Triangulation Genotyping Analysis of Campylobacter Species

A series of triangulation genotyping analysis primers were designed asdescribed in Example 1 with the objective of identification of differentstrains of Campylobacter jejuni. The primers are listed in Table 12 withthe designation “CJST_CJ.” Housekeeping genes to which the primershybridize and produce bioagent identifying amplicons include: tkt(transketolase), glyA (serine hydroxymethyltransferase), gltA (citratesynthase), aspA (aspartate ammonia lyase), glnA (glutamine synthase),pgm (phosphoglycerate mutase), and uncA (ATP synthetase alpha chain).TABLE 12 Campylobacter Genotyping Primer Pairs Primer Pair ForwardPrimer Reverse Primer No. Forward Primer Name (SEQ ID NO:) ReversePrimer Name (SEQ ID NO:) Target Gene 1053 CJST_CJ_1080_1110_F 681CJST_CJ_1166_1198_R 1022 gltA 1047 CJST_CJ_584_616_F 315CJST_CJ_663_692_R 1379 glnA 1048 CJST_CJ_360_394_F 346 CJST_CJ_442_476_R955 aspA 1049 CJST_CJ_2636_2668_F 504 CJST_CJ_2753_2777_R 1409 tkt 1054CJST_CJ_2060_2090_F 323 CJST_CJ_2148_2174_R 1068 pgm 1064CJST_CJ_1680_1713_F 479 CJST_CJ_1795_1822_R 938 glyA

The primers were used to amplify nucleic acid from 50 food productsamples provided by the USDA, 25 of which contained Campylobacter jejuniand 25 of which contained Campylobacter coli. Primers used in this studywere developed primarily for the discrimination of Campylobacter jejuniclonal complexes and for distinguishing Campylobacter jejuni fromCampylobacter coli. Finer discrimination between Campylobacter colitypes is also possible by using specific primers targeted to loci whereclosely-related Campylobacter coli isolates demonstrate polymorphismsbetween strains. The conclusions of the comparison of base compositionanalysis with sequence analysis are shown in Tables 13A-C. TABLE 13AResults of Base Composition Analysis of 50 Campylobacter Samples withDrill-down MLST Primer Pair Nos: 1048 and 1047 Base Base Composition ofComposition of MLST type or Bioagent Bioagent Clonal MLST TypeIdentifying Identifying Complex by or Clonal Amplicon Amplicon BaseComplex by Obtained with Obtained with Isolate Composition SequencePrimer Pair No: Primer Pair Group Species origin analysis analysisStrain 1048 (aspA) No: 1047 (glnA) J-1 C. jejuni Goose ST 690/ ST 991RM3673 A30 G25 C16 T46 A47 G21 C16 T25 692/707/991 J-2 C. jejuni HumanComplex ST 356, RM4192 A30 G25 C16 T46 A48 G21 C17 T23 206/48/353complex 353 J-3 C. jejuni Human Complex ST 436 RM4194 A30 G25 C15 T47A48 G21 C18 T22 354/179 J-4 C. jejuni Human Complex 257 ST 257, RM4197A30 G25 C16 T46 A48 G21 C18 T22 complex 257 J-5 C. jejuni Human Complex52 ST 52, RM4277 A30 G25 C16 T46 A48 G21 C17 T23 complex 52 J-6 C.jejuni Human Complex 443 ST 51, RM4275 A30 G25 C15 T47 A48 G21 C17 T23complex RM4279 A30 G25 C15 T47 A48 G21 C17 T23 443 J-7 C. jejuni HumanComplex 42 ST 604, RM1864 A30 G25 C15 T47 A48 G21 C18 T22 complex 42 J-8C. jejuni Human Complex ST 362, RM3193 A30 G25 C15 T47 A48 G21 C18 T2242/49/362 complex 362 J-9 C. jejuni Human Complex ST 147, RM3203 A30 G25C15 T47 A47 G21 C18 T23 45/283 Complex 45 C. jejuni Human Consistent ST828 RM4183 A31 G27 C20 T39 A48 G21 C16 T24 C-1 C. coli with 74 ST 832RM1169 A31 G27 C20 T39 A48 G21 C16 T24 closely ST 1056 RM1857 A31 G27C20 T39 A48 G21 C16 T24 Poultry related ST 889 RM1166 A31 G27 C20 T39A48 G21 C16 T24 sequence ST 829 RM1182 A31 G27 C20 T39 A48 G21 C16 T24types (none ST 1050 RM1518 A31 G27 C20 T39 A48 G21 C16 T24 belong to aST 1051 RM1521 A31 G27 C20 T39 A48 G21 C16 T24 clonal ST 1053 RM1523 A31G27 C20 T39 A48 G21 C16 T24 complex) ST 1055 RM1527 A31 G27 C20 T39 A48G21 C16 T24 ST 1017 RM1529 A31 G27 C20 T39 A48 G21 C16 T24 ST 860 RM1840A31 G27 C20 T39 A48 G21 C16 T24 ST 1063 RM2219 A31 G27 C20 T39 A48 G21C16 T24 ST 1066 RM2241 A31 G27 C20 T39 A48 G21 C16 T24 ST 1067 RM2243A31 G27 C20 T39 A48 G21 C16 T24 ST 1068 RM2439 A31 G27 C20 T39 A48 G21C16 T24 Swine ST 1016 RM3230 A31 G27 C20 T39 A48 G21 C16 T24 ST 1069RM3231 A31 G27 C20 T39 A48 G21 C16 T24 ST 1061 RM1904 A31 G27 C20 T39A48 G21 C16 T24 Unknown ST 825 RM1534 A31 G27 C20 T39 A48 G21 C16 T24 ST901 RM1505 A31 G27 C20 T39 A48 G21 C16 T24 C-2 C. coli Human ST 895 ST895 RM1532 A31 G27 C19 T40 A48 G21 C16 T24 C-3 C. coli PoultryConsistent ST 1064 RM2223 A31 G27 C20 T39 A48 G21 C16 T24 with 63 ST1082 RM1178 A31 G27 C20 T39 A48 G21 C16 T24 closely ST 1054 RM1525 A31G27 C20 T39 A48 G21 C16 T24 related ST 1049 RM1517 A31 G27 C20 T39 A48G21 C16 T24 Marmoset sequence ST 891 RM1531 A31 G27 C20 T39 A48 G21 C16T24 types (none belong to a clonal complex)

TABLE 13B Results of Base Composition Analysis of 50 CampylobacterSamples with Drill- down MLST Primer Pair Nos: 1053 and 1064 Base BaseComposition of Composition of MLST type or Bioagent Bioagent Clonal MLSTType Identifying Identifying Complex by or Clonal Amplicon Amplicon BaseComplex by Obtained with Obtained with Isolate Composition SequencePrimer Pair Primer Pair Group Species origin analysis analysis StrainNo: 1053 (gltA) No: 1064 (glyA) J-1 C. jejuni Goose ST 690/ ST 991RM3673 A24 G25 C23 T47 A40 G29 C29 T45 692/707/991 J-2 C. jejuni HumanComplex ST 356, RM4192 A24 G25 C23 T47 A40 G29 C29 T45 206/48/353complex 353 J-3 C. jejuni Human Complex ST 436 RM4194 A24 G25 C23 T47A40 G29 C29 T45 354/179 J-4 C. jejuni Human Complex 257 ST 257, RM4197A24 G25 C23 T47 A40 G29 C29 T45 complex 257 J-5 C. jejuni Human Complex52 ST 52, RM4277 A24 G25 C23 T47 A39 G30 C26 T48 complex 52 J-6 C.jejuni Human Complex 443 ST 51, RM4275 A24 G25 C23 T47 A39 G30 C28 T46complex RM4279 A24 G25 C23 T47 A39 G30 C28 T46 443 J-7 C. jejuni HumanComplex 42 ST 604, RM1864 A24 G25 C23 T47 A39 G30 C26 T48 complex 42 J-8C. jejuni Human Complex ST 362, RM3193 A24 G25 C23 T47 A38 G31 C28 T4642/49/362 complex 362 J-9 C. jejuni Human Complex ST 147, RM3203 A24 G25C23 T47 A38 G31 C28 T46 45/283 Complex 45 C. jejuni Human Consistent ST828 RM4183 A23 G24 C26 T46 A39 G30 C27 T47 C-1 C. coli with 74 ST 832RM1169 A23 G24 C26 T46 A39 G30 C27 T47 closely ST 1056 RM1857 A23 G24C26 T46 A39 G30 C27 T47 Poultry related ST 889 RM1166 A23 G24 C26 T46A39 G30 C27 T47 sequence ST 829 RM1182 A23 G24 C26 T46 A39 G30 C27 T47types (none ST 1050 RM1518 A23 G24 C26 T46 A39 G30 C27 T47 belong to aST 1051 RM1521 A23 G24 C26 T46 A39 G30 C27 T47 clonal ST 1053 RM1523 A23G24 C26 T46 A39 G30 C27 T47 complex) ST 1055 RM1527 A23 G24 C26 T46 A39G30 C27 T47 ST 1017 RM1529 A23 G24 C26 T46 A39 G30 C27 T47 ST 860 RM1840A23 G24 C26 T46 A39 G30 C27 T47 ST 1063 RM2219 A23 G24 C26 T46 A39 G30C27 T47 ST 1066 RM2241 A23 G24 C26 T46 A39 G30 C27 T47 ST 1067 RM2243A23 G24 C26 T46 A39 G30 C27 T47 ST 1068 RM2439 A23 G24 C26 T46 A39 G30C27 T47 Swine ST 1016 RM3230 A23 G24 C26 T46 A39 G30 C27 T47 ST 1069RM3231 A23 G24 C26 T46 NO DATA ST 1061 RM1904 A23 G24 C26 T46 A39 G30C27 T47 Unknown ST 825 RM1534 A23 G24 C26 T46 A39 G30 C27 T47 ST 901RM1505 A23 G24 C26 T46 A39 G30 C27 T47 C-2 C. coli Human ST 895 ST 895RM1532 A23 G24 C26 T46 A39 G30 C27 T47 C-3 C. coli Poultry Consistent ST1064 RM2223 A23 G24 C26 T46 A39 G30 C27 T47 with 63 ST 1082 RM1178 A23G24 C26 T46 A39 G30 C27 T47 closely ST 1054 RM1525 A23 G24 C25 T47 A39G30 C27 T47 related ST 1049 RM1517 A23 G24 C26 T46 A39 G30 C27 T47Marmoset sequence ST 891 RM1531 A23 G24 C26 T46 A39 G30 C27 T47 types(none belong to a clonal complex)

TABLE 13C Results of Base Composition Analysis of 50 CampylobacterSamples with Drill- down MLST Primer Pair Nos: 1054 and 1049 Base BaseComposition of Composition of MLST type or Bioagent Bioagent Clonal MLSTType Identifying Identifying Complex by or Clonal Amplicon Amplicon BaseComplex by Obtained with Obtained with Isolate Composition SequencePrimer Pair No: Primer Pair Group Species origin analysis analysisStrain 1054 (pgm) No: 1049 (tkt) J-1 C. jejuni Goose ST 690/ ST 991RM3673 A26 G33 C18 T38 A41 G28 C35 T38 692/707/991 J-2 C. jejuni HumanComplex ST 356, RM4192 A26 G33 C19 T37 A41 G28 C36 T37 206/48/353complex 353 J-3 C. jejuni Human Complex ST 436 RM4194 A27 G32 C19 T37A42 G28 C36 T36 354/179 J-4 C. jejuni Human Complex 257 ST 257, RM4197A27 G32 C19 T37 A41 G29 C35 T37 complex 257 J-5 C. jejuni Human Complex52 ST 52, RM4277 A26 G33 C18 T38 A41 G28 C36 T37 complex 52 J-6 C.jejuni Human Complex 443 ST 51, RM4275 A27 G31 C19 T38 A41 G28 C36 T37complex RM4279 A27 G31 C19 T38 A41 G28 C36 T37 443 J-7 C. jejuni HumanComplex 42 ST 604, RM1864 A27 G32 C19 T37 A42 G28 C35 T37 complex 42 J-8C. jejuni Human Complex ST 362, RM3193 A26 G33 C19 T37 A42 G28 C35 T3742/49/362 complex 362 J-9 C. jejuni Human Complex ST 147, RM3203 A28 G31C19 T37 A43 G28 C36 T35 45/283 Complex 45 C. jejuni Human Consistent ST828 RM4183 A27 G30 C19 T39 A46 G28 C32 T36 C-1 C. coli with 74 ST 832RM1169 A27 G30 C19 T39 A46 G28 C32 T36 closely ST 1056 RM1857 A27 G30C19 T39 A46 G28 C32 T36 Poultry related ST 889 RM1166 A27 G30 C19 T39A46 G28 C32 T36 sequence ST 829 RM1182 A27 G30 C19 T39 A46 G28 C32 T36types (none ST 1050 RM1518 A27 G30 C19 T39 A46 G28 C32 T36 belong to aST 1051 RM1521 A27 G30 C19 T39 A46 G28 C32 T36 clonal ST 1053 RM1523 A27G30 C19 T39 A46 G28 C32 T36 complex) ST 1055 RM1527 A27 G30 C19 T39 A46G28 C32 T36 ST 1017 RM1529 A27 G30 C19 T39 A46 G28 C32 T36 ST 860 RM1840A27 G30 C19 T39 A46 G28 C32 T36 ST 1063 RM2219 A27 G30 C19 T39 A46 G28C32 T36 ST 1066 RM2241 A27 G30 C19 T39 A46 G28 C32 T36 ST 1067 RM2243A27 G30 C19 T39 A46 G28 C32 T36 ST 1068 RM2439 A27 G30 C19 T39 A46 G28C32 T36 Swine ST 1016 RM3230 A27 G30 C19 T39 A46 G28 C32 T36 ST 1069RM3231 A27 G30 C19 T39 A46 G28 C32 T36 ST 1061 RM1904 A27 G30 C19 T39A46 G28 C32 T36 Unknown ST 825 RM1534 A27 G30 C19 T39 A46 G28 C32 T36 ST901 RM1505 A27 G30 C19 T39 A46 G28 C32 T36 C-2 C. coli Human ST 895 ST895 RM1532 A27 G30 C19 T39 A45 G29 C32 T36 C-3 C. coli PoultryConsistent ST 1064 RM2223 A27 G30 C19 T39 A45 G29 C32 T36 with 63 ST1082 RM1178 A27 G30 C19 T39 A45 G29 C32 T36 closely ST 1054 RM1525 A27G30 C19 T39 A45 G29 C32 T36 related ST 1049 RM1517 A27 G30 C19 T39 A45G29 C32 T36 Marmoset sequence ST 891 RM1531 A27 G30 C19 T39 A45 G29 C32T36 types (none belong to a clonal complex)

The base composition analysis method was successful in identification of12 different strain groups. Campylobacter jejuni and Campylobacter coliare generally differentiated by all loci. Ten clearly differentiatedCampylobacter jejuni isolates and 2 major Campylobacter coli groups wereidentified even though the primers were designed for strain typing ofCampylobacter jejuni. One isolate (RM4183) which was designated asCampylobacter jejuni was found to group with Campylobacter coli and alsoappears to actually be Campylobacter coli by full MLST sequencing.

Example 11 Identification of Acinetobacter baumannii Using Broad RangeSurvey and Division-Wide Primers in Epidemiological Surveillance

To test the capability of the broad range survey and division-wideprimer sets of Table 5 in identification of Acinetobacter species, 183clinical samples were obtained from individuals participating in, or incontact with individuals participating in Operation Iraqi Freedom(including US service personnel, US civilian patients at the Walter ReedArmy Institute of Research (WRAIR), medical staff, Iraqi civilians andenemy prisoners. In addition, 34 environmental samples were obtainedfrom hospitals in Iraq, Kuwait, Germany, the United States and the USNSComfort, a hospital ship.

Upon amplification of nucleic acid obtained from the clinical samples,primer pairs 346-349, 360, 361, 354, 362 and 363 (Table 5) all producedbacterial bioagent amplicons which identified Acinetobacter baumannii in215 of 217 samples. The organism Klebsiella pneumoniae was identified inthe remaining two samples. In addition, 14 different strain types(containing single nucleotide polymorphisms relative to a referencestrain of Acinetobacter baumannii) were identified and assignedarbitrary numbers from 1 to 14. Strain type 1 was found in 134 of thesample isolates and strains 3 and 7 were found in 46 and 9 of theisolates respectively.

The epidemiology of strain type 7 of Acinetobacter baumannii wasinvestigated. Strain 7 was found in 4 patients and 5 environmentalsamples (from field hospitals in Iraq and Kuwait). The index patientinfected with strain 7 was a pre-war patient who had a traumaticamputation in March of 2003 and was treated at a Kuwaiti hospital. Thepatient was subsequently transferred to a hospital in Germany and thento WRAIR. Two other patients from Kuwait infected with strain 7 werefound to be non-infectious and were not further monitored. The fourthpatient was diagnosed with a strain 7 infection in September of 2003 atWRAIR. Since the fourth patient was not related involved in OperationIraqi Freedom, it was inferred that the fourth patient was the subjectof a nosocomial infection acquired at WRAIR as a result of the spread ofstrain 7 from the index patient.

The epidemiology of strain type 3 of Acinetobacter baumannii was alsoinvestigated. Strain type 3 was found in 46 samples, all of which werefrom patients (US service members, Iraqi civilians and enemy prisoners)who were treated on the USNS Comfort hospital ship and subsequentlyreturned to Iraq or Kuwait. The occurrence of strain type 3 in a singlelocale may provide evidence that at least some of the infections at thatlocale were a result of nosocomial infections.

This example thus illustrates an embodiment of the present inventionwherein the methods of analysis of bacterial bioagent identifyingamplicons provide the means for epidemiological surveillance.

Example 12 Selection and Use of Triangulation Genotyping Analysis PrimerPairs for Acinetobacter baumanii

To combine the power of high-throughput mass spectrometric analysis ofbioagent identifying amplicons with the sub-species characteristicresolving power provided by triangulation genotyping analysis, anadditional 21 primer pairs were selected based on analysis ofhousekeeping genes of the genus Acinetobacter. Genes to which thedrill-down triangulation genotyping analysis primers hybridize forproduction of bacterial bioagent identifying amplicons includeanthranilate synthase component I (trpE), adenylate kinase (adk),adenine glycosylase (mutY), fumarate hydratase (fumC), and pyrophosphatephospho-hydratase (ppa). These 21 primer pairs are indicated withreference to sequence listings in Table 14. Primer pair numbers1151-1154 hybridize to and amplify segments of trpE. Primer pair numbers1155-1157 hybridize to and amplify segments of adk. Primer pair numbers1158-1164 hybridize to and amplify segments of mutY. Primer pair numbers1165-1170 hybridize to and amplify segments of fumC. Primer pair number1171 hybridizes to and amplifies a segment of ppa. Primer pair numbers:2846-2848 hybridize to and amplify segments of the parC gene of DNAtopoisomerase which include a codon known to confer quinolone drugresistance upon sub-types of Acinetobacter baumannii. Primer pairnumbers 2852-2854 hybridize to and amplify segments of the gyrA gene ofDNA gyrase which include a codon known to confer quinolone drugresistance upon sub-types of Acinetobacter baumannii. Primer pairnumbers 2922 and 2972 are speciating primers which are useful foridentifying different species members of the genus Acinetobacter. Theprimer names given in Table 14A (with the exception of primer pairnumbers 2846-2848, 2852-2854) indicate the coordinates to which theprimers hybridize to a reference sequence which comprises aconcatenation of the genes TrpE, efp (elongation factor p), adk, mutT,fumC, and ppa. For example, the forward primer of primer pair 1151 isnamed AB_MLST-11-OIF007_(—)62_(—)91_F because it hybridizes to theAcinetobacter primer reference sequence of strain type 11 in sample 007of Operation Iraqi Freedom (OIF) at positions 62 to 91. DNA wassequenced from strain type 11 and from this sequence data and anartificial concatenated sequence of partial gene extractions wasassembled for use in design of the triangulation genotyping analysisprimers. The stretches of arbitrary residues “N”s in the concatenatedsequence were added for the convenience of separation of the partialgene extractions (40N for AB_MLST (SEQ ID NO: 1444)).

The hybridization coordinates of primer pair numbers 2846-2848 are withrespect to GenBank Accession number X95819. The hybridizationcoordinates of primer pair numbers 2852-2854 are with respect to GenBankAccession number AY642140. Sequence residue “I” appearing in the forwardand reverse primers of primer pair number 2972 represents inosine. TABLE14A Triangulation Genotyping Analysis Primer Pairs for Identification ofSub-species characteristics (Strain Type) of Members of the BacterialGenus Acinetobacter Primer Forward Primer Reverse Primer Pair No.Forward Primer Name (SEQ ID NO:) Reverse Primer Name (SEQ ID NO:) 1151AB_MLST-11-OIF007_62_91_F 454 AB_MLST-11-OIF007_169_203_R 1418 1152AB_MLST-11-OIF007_185_214_F 243 AB_MLST-11-OIF007_291_324_R 969 1153AB_MLST-11-OIF007_260_289_F 541 AB_MLST-11-OIF007_364_393_R 1400 1154AB_MLST-11-OIF007_206_239_F 436 AB_MLST-11-OIF007_318_344_R 1036 1155AB_MLST-11-OIF007_522_552_F 378 AB_MLST-11-OIF007_587_610_R 1392 1156AB_MLST-11-OIF007_547_571_F 250 AB_MLST-11-OIF007_656_686_R 902 1157AB_MLST-11-OIF007_601_627_F 256 AB_MLST-11-OIF007_710_736_R 881 1158AB_MLST-11-OIF007_1202_1225_F 384 AB_MLST-11-OIF007_1266_1296_R 878 1159AB_MLST-11-OIF007_1202_1225_F 384 AB_MLST-11-OIF007_1299_1316_R 11991160 AB_MLST-11-OIF007_1234_1264_F 694 AB_MLST-11-OIF007_1335_1362_R1215 1161 AB_MLST-11-OIF007_1327_1356_F 225AB_MLST-11-OIF007_1422_1448_R 1212 1162 AB_MLST-11-OIF007_1345_1369_F383 AB_MLST-11-OIF007_1470_1494_R 1083 1163AB_MLST-11-OIF007_1351_1375_F 662 AB_MLST-11-OIF007_1470_1494_R 10831164 AB_MLST-11-OIF007_1387_1412_F 422 AB_MLST-11-OIF007_1470_1494_R1083 1165 AB_MLST-11-OIF007_1542_1569_F 194AB_MLST-11-OIF007_1656_1680_R 1173 1166 AB_MLST-11-OIF007_1566_1593_F684 AB_MLST-11-OIF007_1656_1680_R 1173 1167AB_MLST-11-OIF007_1611_1638_F 375 AB_MLST-11-OIF007_1731_1757_R 890 1168AB_MLST-11-OIF007_1726_1752_F 182 AB_MLST-11-OIF007_1790_1821_R 11951169 AB_MLST-11-OIF007_1792_1826_F 656 AB_MLST-11-OIF007_1876_1909_R1151 1170 AB_MLST-11-OIF007_1792_1826_F 656AB_MLST-11-OIF007_1895_1927_R 1224 1171 AB_MLST-11-OIF007_1970_2002_F618 AB_MLST-11-OIF007_2097_2118_R 1157 2846 PARC_X95819_33_58_F 302PARC_X95819_121_153_R 852 2847 PARC_X95819_33_58_F 199PARC_X95819_157_178_R 889 2848 PARC_X95819_33_58_F 596PARC_X95819_97_128_R 1169 2852 GYRA_AY642140_−1_24_F 150GYRA_AY642140_71_100_R 1242 2853 GYRA_AY642140_26_54_F 166GYRA_AY642140_121_146_R 1069 2854 GYRA_AY642140_26_54_F 166GYRA_AY642140_58_89_R 1168 2922 AB_MLST-11-OIF007_991_1018_F 583AB_MLST-11-OIF007_1110_1137_R 923 2972 AB_MLST-11-OIF007_1007_1034_F 592AB_MLST-11-OIF007_1126_1153_R 924

TABLE 14B Triangulation Genotyping Analysis Primer Pairs forIdentification of Sub-species characteristics (Strain Type) of Membersof the Bacterial Genus Acinetobacter Primer Reverse Primer ForwardPrimer Pair (SEQ ID (SEQ ID No. NO:) SEQUENCE NO:) SEQUENCE 1151 454TGAGATTGCTGAACATTTAATGCTGATTGA 1418 TTGTACATTTGAAACAATATGCATGACATGTGAAT1152 243 TATTGTTTCAAATGTACAAGGTGAAGTGCG 969TCACAGGTTCTACTTCATCAATAATTTCCATTGC 1153 541TGGAACGTTATCAGGTGCCCCAAAAATTCG 1400 TTGCAATCGACATATCCATTTCACCATGCC 1154436 TGAAGTGCGTGATGATATCGATGCACTTGATGTA 1036 TCCGGCAAAAACTCCCCTTTTCACAGG1155 378 TCGGTTTAGTAAAAGAACGTATTGCTCAACC 1392 TTCTGCTTGAGGAATAGTGCGTGG1156 250 TCAACCTGACTGCGTGAATGGTTGT 902 TACGTTCTACGATTTCTTCATCAGGTACATC1157 256 TCAAGCAGAAGCTTTGGAAGAAGAAGG 881 TACAACGTGATAAACACGACCAGAAGC1158 384 TCGTGCCCGCAATTTGCATAAAGC 878 TAATGCCGGGTAGTGCAATCCATTCTTCTAG1159 384 TCGTGCCCGCAATTTGCATAAAGC 1199 TGCACCTGCGGTCGAGCG 1160 694TTGTAGCACAGCAAGGCAAATTTCCTGAAAC 1215 TGCCATCCATAATCACGCCATACTGACG 1161225 TAGGTTTACGTCAGTATGGCGTGATTATGG 1212 TGCCAGTTTCCACATTTCACGTTCGTG 1162383 TCGTGATTATGGATGGCAACGTGAA 1083 TCGCTTGAGTGTAGTCATGATTGCG 1163 662TTATGGATGGCAACGTGAAACGCGT 1083 TCGCTTGAGTGTAGTCATGATTGCG 1164 422TCTTTGCCATTGAAGATGACTTAAGC 1083 TCGCTTGAGTGTAGTCATGATTGCG 1165 194TACTAGCGGTAAGCTTAAACAAGATTGC 1173 TGAGTCGGGTTCACTTTACCTGGCA 1166 684TTGCCAATGATATTCGTTGGTTAGCAAG 1173 TGAGTCGGGTTCACTTTACCTGGCA 1167 375TCGGCGAAATCCGTATTCCTGAAAATGA 890 TACCGGAAGCACCAGCGACATTAATAG 1168 182TACCACTATTAATGTCGCTGGTGCTTC 1195 TGCAACTGAATAGATTGCAGTAAGTTATAAGC 1169656 TTATAACTTACTGCAATCTATTCAGTTGCTTGGTG 1151TGAATTATGCAAGAAGTGATCAATTTTCTCACGA 1170 656TTATAACTTACTGCAATCTATTCAGTTGCTTGGTG 1224TGCCGTAACTAACATAAGAGAATTATGCAAGAA 1171 618TGGTTATGTACCAAATACTTTGTCTGAAGATGG 1157 TGACGGCATCGATACCACCGTC 2846 302TCCAAAAAAATCAGCGCGTACAGTGG 852 TAAAGGATAGCGGTAACTAAATGGCTGAGCCAT 2847199 TACTTGGTAAATACCACCCACATGGTGA 889 TACCCCAGTTCCCCTGACCTTC 2848 596TGGTAAATACCACCCACATGGTGAC 1169 TGAGCCATGAGTACCATGGCTTCATAACATGC 2852 150TAAATCTGCCCGTGTCGTTGGTGAC 1242 TGCTAAAGTCTTGAGCCATACGAACAATGG 2853 166TAATCGGTAAATATCACCCGCATGGTGAC 1069 TCGATCGAACCGAAGTTACCCTGACC 2854 166TAATCGGTAAATATCACCCGCATGGTGAC 1168 TGAGCCATACGAACAATGGTTTCATAAACAGC 2922583 TGGGCGATGCTGCGAAATGGTTAAAAGA 923 TAGTATCACCACGTACACCCGGATCAGT 2972592 TGGGIGATGCTGCIAAATGGTTAAAAGA 924 TAGTATCACCACGTACICCIGGATCAGT

Analysis of bioagent identifying amplicons obtained using the primers ofTable 14B for over 200 samples from Operation Iraqi Freedom resulted inthe identification of 50 distinct strain type clusters. The largestcluster, designated strain type 11 (ST11) includes 42 sample isolates,all of which were obtained from US service personnel and Iraqi civilianstreated at the 28^(th) Combat Support Hospital in Baghdad. Several ofthese individuals were also treated on the hospital ship USNS Comfort.These observations are indicative of significant epidemiologicalcorrelation/linkage.

All of the sample isolates were tested against a broad panel ofantibiotics to characterize their antibiotic resistance profiles. As anexample of a representative result from antibiotic susceptibilitytesting, ST11 as found to consist of four different clusters ofisolates, each with a varying degree of sensitivity/resistance to thevarious antibiotics tested which included penicillins, extended spectrumpenicillins, cephalosporins, carbepenem, protein synthesis inhibitors,nucleic acid synthesis inhibitors, anti-metabolites, and anti-cellmembrane antibiotics. Thus, the genotyping power of bacterial bioagentidentifying amplicons, particularly drill-down bacterial bioagentidentifying amplicons, has the potential to increase the understandingof the transmission of infections in combat casualties, to identify thesource of infection in the environment, to track hospital transmissionof nosocomial infections, and to rapidly characterize drug-resistanceprofiles which enable development of effective infection controlmeasures on a time-scale previously not achievable.

Example 13 Triangulation Genotyping Analysis and Codon Analysis ofAcinetobacter baumannii Samples from Two Health Care Facilities

In this investigation, 88 clinical samples were obtained from WalterReed Hospital and 95 clinical samples were obtained from NorthwesternMedical Center. All samples from both healthcare facilities weresuspected of containing sub-types of Acinetobacter baumannii, at leastsome of which were expected to be resistant to quinolone drugs. Each ofthe 183 samples was analyzed by the method of the present invention. DNAwas extracted from each of the samples and amplified with eighttriangulation genotyping analysis primer pairs represented by primerpair numbers: 1151, 1156, 1158, 1160, 1165, 1167, 1170, and 1171. TheDNA was also amplified with speciating primer pair number 2922 and codonanalysis primer pair numbers 2846-2848 which interrogate a codon presentin the parC gene, and primer pair numbers 2852-2854 which bracket acodon present in the gyrA gene. The parC and gyrA codon mutations areboth responsible for causing drug resistance in Acinetobacter baumannii.During evolution of drug resistant strains, the gyrA mutation usuallyoccurs before the parC mutation. Amplification products were measured byESI-TOF mass spectrometry as indicated in Example 4. The basecompositions of the amplification products were calculated from theaverage molecular masses of the amplification products and are shown inTables 15-18. The entries in each of the tables are grouped according tostrain type number, which is an arbitrary number assigned toAcinetobacter baumannii strains in the order of observance beginningfrom the triangulation genotyping analysis OIF genotyping studydescribed in Example 12. For example, strain type 11 which appears insamples from the Walter Reed Hospital is the same strain as the straintype 11 mentioned in Example 12. Ibis# refers to the order in which eachsample was analyzed. Isolate refers to the original sample isolatenumbering system used at the location from which the samples wereobtained (either Walter Reed Hospital or Northwestern Medical Center).ST=strain type. ND=not detected. Base compositions highlighted with boldtype indicate that the base composition is a unique base composition forthe amplification product obtained with the pair of primers indicated.TABLE 15A Base Compositions of Amplification Products of 88 A. baumanniiSamples Obtained from Walter Reed Hospital and Amplified with CodonAnalysis Primer Pairs Targeting the gyrA Gene PP No: 2852 PP No: 2853 PPNo: 2854 Species Ibis# Isolate ST gyrA gyrA gyrA A. baumannii 20 1082 1A25G23C22T31 A29G28C22T42 A17G13C14T20 A. baumannii 13  854 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 22 1162 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 27 1230 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 31 1367 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 37 1459 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 55 1700 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 64 1777 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 73 1861 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 74 1877 10 NDA29G28C21T43 A17G13C13T21 A. baumannii 86 1972 10 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  3  684 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  6  720 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  7  726 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 19 1079 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 21 1123 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 23 1188 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 33 1417 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 34 1431 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 38 1496 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 40 1523 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 42 1640 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 50 1666 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 51 1668 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 52 1695 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 65 1781 11 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 44 1649 12 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii   49A   1658.1 12 A25G23C22T31A29G28C21T43 A17G13C13T21 A. baumannii   49B   1658.2 12 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 56 1707 12 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 80 1893 12 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  5  693 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  8  749 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 10  839 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 14  865 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 16  888 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 29 1326 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 35 1440 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 41 1524 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 46 1652 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 47 1653 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 48 1657 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 57 1709 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 61 1727 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 63 1762 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 67 1806 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 75 1881 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 77 1886 14 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  1  649 46 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  2  653 46 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 39 1497 16 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 24 1198 15 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 28 1243 15 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 43 1648 15 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 62 1746 15 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii  4  689 15 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 68 1822 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 69   1823A 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 70   1823B 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 71 1826 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 72 1860 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 81 1924 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 82 1929 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 85 1966 3 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 11  841 3 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii 32 1415 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 45 1651 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 54 1697 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 58 1712 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 60 1725 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 66 1802 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 76 1883 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 78 1891 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 79 1892 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 83 1947 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 84 1964 24 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 53 1696 24 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii 36 1458 49 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 59 1716 9 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii  9  805 30 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii 18  967 39 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii 30 1322 48 A25G23C22T31A29G28C22T42 A17G13C14T20 A. baumannii 26 1218 50 A25G23C22T31A29G28C22T42 A17G13C14T20 A. sp. 13TU 15  875 A1 A25G23C22T31A29G28C22T42 A17G13C14T20 A. sp. 13TU 17  895 A1 A25G23C22T31A29G28C22T42 A17G13C14T20 A. sp. 3 12  853 B7 A25G22C22T32 A30G29C22T40A17G13C14T20 A. johnsonii 25 1202 NEW1 A25G22C22T32 A30G29C22T40A17G13C14T20 A. sp. 2082 87 2082 NEW2 A25G22C22T32 A31G28C22T40A17G13C14T20

TABLE 15B Base Compositions Determined from A. baumannii DNA SamplesObtained from Walter Reed Hospital and Amplified with Codon AnalysisPrimer Pairs Targeting the parC Gene PP No: 2846 PP No: 2847 PP No: 2848Species Ibis# Isolate ST parC parC parC A. baumannii 20 1082 1A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 13  854 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 22 1162 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 27 1230 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 31 1367 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 37 1459 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 55 1700 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 64 1777 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 73 1861 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 74 1877 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 86 1972 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  3  684 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  6  720 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  7  726 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 19 1079 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 21 1123 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 23 1188 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 33 1417 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 34 1431 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 38 1496 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 40 1523 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 42 1640 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 50 1666 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 51 1668 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 52 1695 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 65 1781 11A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 44 1649 12A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii   49A   1658.1 12A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii   49B   1658.2 12A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 56 1707 12A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 80 1893 12A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  5  693 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  8  749 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 10  839 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 14  865 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 16  888 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 29 1326 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 35 1440 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 41 1524 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 46 1652 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 47 1653 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 48 1657 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 57 1709 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 61 1727 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 63 1762 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 67 1806 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 75 1881 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 77 1886 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  1  649 46A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii  2  653 46A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 39 1497 16A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 24 1198 15A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii 28 1243 15A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii 43 1648 15A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii 62 1746 15A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii  4  689 15A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 68 1822 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 69   1823A 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 70   1823B 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 71 1826 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 72 1860 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 81 1924 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 82 1929 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 85 1966 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 11  841 3A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 32 1415 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 45 1651 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 54 1697 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 58 1712 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 60 1725 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 66 1802 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 76 1883 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 78 1891 24A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 79 1892 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 83 1947 24A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 84 1964 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 53 1696 24A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 36 1458 49A34G26C29T32 A30G28C24T32 A16G14C15T15 A. baumannii 59 1716 9A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii  9  805 30A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 18  967 39A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 30 1322 48A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 26 1218 50A33G26C29T33 A29G28C26T31 A16G14C15T15 A. sp. 13TU 15  875 A1A32G26C28T35 A28G28C24T34 A16G14C15T15 A. sp. 13TU 17  895 A1A32G26C28T35 A28G28C24T34 A16G14C15T15 A. sp. 3 12  853 B7 A29G26C27T39A26G32C21T35 A16G14C15T15 A. johnsonii 25 1202 NEW1 A32G28C26T35A29G29C22T34 A16G14C15T15 A. sp. 2082 87 2082 NEW2 A33G27C26T35A31G28C20T35 A16G14C15T15

TABLE 16A Base Compositions Determined from A. baumannii DNA SamplesObtained from Northwestern Medical Center and Amplified with CodonAnalysis Primer Pairs Targeting the gyrA Gene PP No: 2852 PP No: 2853 PPNo: 2854 Species Ibis# Isolate ST gyrA gyrA gyrA A. baumannii 54 536 3A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 87 665 3A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 8 80 10 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 9 91 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 10 92 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 11 131 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 12 137 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 21 218 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 26 242 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 94 678 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 1 9 10 A25G23C21T32 A29G28C21T43 A17G13C13T21A. baumannii 2 13 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii3 19 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 4 24 10A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 5 36 10 A25G23C21T32A29G28C21T43 A17G13C13T21 A. baumannii 6 39 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 13 139 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 15 165 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 16 170 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 17 186 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 20 202 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 22 221 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 24 234 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 25 239 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 33 370 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 34 389 10 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 19 201 14 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 27 257 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 29 301 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 31 354 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 36 422 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 37 424 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 38 434 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 39 473 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 40 482 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 44 512 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 45 516 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 47 522 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 48 526 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 50 528 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 52 531 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 53 533 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 56 542 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 59 550 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 62 556 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 64 557 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 70 588 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 73 603 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 74 605 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 75 606 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 77 611 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 79 622 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 83 643 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 85 653 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 89 669 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 93 674 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 23 228 51 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 32 369 52 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 35 393 52 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 30 339 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 41 485 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 42 493 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 43 502 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 46 520 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 49 527 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 51 529 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 65 562 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 68 579 53 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 57 546 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 58 548 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 60 552 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 61 555 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 63 557 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 66 570 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 67 578 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 69 584 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 71 593 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 72 602 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 76 609 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 78 621 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 80 625 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 81 628 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 82 632 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 84 649 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 86 655 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 88 668 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 90 671 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 91 672 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 92 673 54 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 18 196 55 A25G23C22T31 A29G28C21T43A17G13C13T21 A. baumannii 55 537 27 A25G23C21T32 A29G28C21T43A17G13C13T21 A. baumannii 28 263 27 A25G23C22T31 A29G28C22T42A17G13C14T20 A. sp. 3 14 164 B7 A25G22C22T32 A30G29C22T40 A17G13C14T20mixture 7 71 — ND ND A17G13C15T19

TABLE 16B Base Compositions Determined from A. baumannii DNA SamplesObtained from Northwestern Medical Center and Amplified with CodonAnalysis Primer Pairs Targeting the parC Gene PP No: 2846 PP No: 2847 PPNo: 2848 Species Ibis# Isolate ST parC parC parC A. baumannii 54 536 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 87 665 3A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 8 80 10 A33G26C28T34A29G28C25T32 A16G14C14T16 A. baumannii 9 91 10 A33G26C28T34 A29G28C25T32A16G14C14T16 A. baumannii 10 92 10 A33G26C28T34 A29G28C25T32 ND A.baumannii 11 131 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii12 137 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 21 218 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 26 242 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 94 678 10A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 1 9 10 A33G26C29T33A29G28C26T31 A16G14C15T15 A. baumannii 2 13 10 A33G26C29T33 A29G28C26T31A16G14C15T15 A. baumannii 3 19 10 A33G26C29T33 A29G28C26T31 A16G14C15T15A. baumannii 4 24 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii5 36 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 6 39 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 13 139 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 15 165 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 16 170 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 17 186 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 20 202 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 22 221 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 24 234 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 25 239 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 33 370 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 34 389 10A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 19 201 14A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 27 257 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 29 301 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 31 354 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 36 422 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 37 424 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 38 434 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 39 473 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 40 482 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 44 512 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 45 516 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 47 522 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 48 526 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 50 528 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 52 531 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 53 533 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 56 542 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 59 550 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 62 556 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 64 557 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 70 588 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 73 603 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 74 605 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 75 606 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 77 611 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 79 622 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 83 643 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 85 653 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 89 669 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 93 674 51A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 23 228 51A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 32 369 52A34G25C28T34 A30G27C25T32 A16G14C14T16 A. baumannii 35 393 52A34G25C28T34 A30G27C25T32 A16G14C14T16 A. baumannii 30 339 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 41 485 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 42 493 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 43 502 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 46 520 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 49 527 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 51 529 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 65 562 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 68 579 53A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 57 546 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 58 548 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 60 552 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 61 555 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 63 557 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 66 570 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 67 578 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 69 584 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 71 593 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 72 602 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 76 609 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 78 621 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 80 625 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 81 628 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 82 632 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 84 649 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 86 655 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 88 668 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 90 671 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 91 672 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 92 673 54A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 18 196 55A33G27C28T33 A29G28C25T31 A15G14C15T16 A. baumannii 55 537 27A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 28 263 27A33G26C29T33 A29G28C26T31 A16G14C15T15 A. sp. 3 14 164 B7 A35G25C29T32A30G28C17T39 A16G14C15T15 mixture 7 71 — ND ND A17G14C15T14

TABLE 17A Base Compositions Determined from A. baumannii DNA SamplesObtained from Walter Reed Hospital and Amplified with Speciating PrimerPair No. 2922 and Triangulation Genotyping Analysis Primer Pair Nos.1151 and 1156 PP No: 2922 PP No: 1151 PP No: 1156 Species Ibis# IsolateST efp trpE Adk A. baumannii 20 1082 1 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 13  854 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 22 1162 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 27 1230 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 31 1367 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 37 1459 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 55 1700 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 64 1777 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 73 1861 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 74 1877 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 86 1972 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii  3  684 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii  6  720 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii  7  726 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 19 1079 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 21 1123 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 23 1188 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 33 1417 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 34 1431 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 38 1496 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 40 1523 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 42 1640 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 50 1666 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 51 1668 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 52 1695 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 65 1781 11 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 44 1649 12 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 49A 1658.1 12 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 49B 1658.2 12 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 56 1707 12 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 80 1893 12 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii  5  693 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii  8  749 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii 10  839 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii 14  865 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii 16  888 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii 29 1326 14 A44G35C25T43 A44G35C22T41A44G32C27T37 A. baumannii 35 1440 14 A44G35C25T43 ND A44G32C27T37 A.baumannii 41 1524 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii46 1652 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 47 165314 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 48 1657 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 57 1709 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 61 1727 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 63 1762 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 67 1806 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 75 1881 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 77 1886 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii  1  649 46A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii  2  653 46A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 39 1497 16A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 24 1198 15A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 28 1243 15A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 43 1648 15A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 62 1746 15A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii  4  689 15A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 68 1822 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 69 1823A 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 70 1823B 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 71 1826 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 72 1860 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 81 1924 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 82 1929 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 85 1966 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 11  841 3A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 32 1415 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 45 1651 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 54 1697 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 58 1712 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 60 1725 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 66 1802 24A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 76 1883 24 NDA43G36C20T43 A44G32C27T37 A. baumannii 78 1891 24 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 79 1892 24 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 83 1947 24 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 84 1964 24 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 53 1696 24 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 36 1458 49 A44G35C25T43A44G35C22T41 A44G32C27T37 A. baumannii 59 1716 9 A44G35C25T43A44G35C21T42 A44G32C26T38 A. baumannii  9  805 30 A44G35C25T43A44G35C19T44 A44G32C27T37 A. baumannii 18  967 39 A45G34C25T43A44G35C22T41 A44G32C26T38 A. baumannii 30 1322 48 A44G35C25T43A43G36C20T43 A44G32C27T37 A. baumannii 26 1218 50 A44G35C25T43A44G35C21T42 A44G32C26T38 A. sp. 13TU 15  875 A1 A47G33C24T43A46G32C20T44 A44G33C27T36 A. sp. 13TU 17  895 A1 A47G33C24T43A46G32C20T44 A44G33C27T36 A. sp. 3 12  853 B7 A46G35C24T42 A42G34C20T46A43G33C24T40 A. johnsonii 25 1202 NEW1 A46G35C23T43 A42G35C21T44A43G33C23T41 A. sp. 2082 87 2082 NEW2 A46G36C22T43 A42G32C20T48A42G34C23T41

TABLE 17B Base Compositions Determined from A. baumannii DNA SamplesObtained from Walter Reed Hospital and Amplified with TriangulationGenotyping Analysis Primer Pair Nos. 1158 and 1160 and 1165 PP No: 1158PP No: 1160 PP No: 1165 Species Ibis# Isolate ST mutY mutY fumC A.baumannii 20 1082 1 A27G21C25T22 A32G35C29T33 A40G33C30T36 A. baumannii13  854 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 22 116210 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 27 1230 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 31 1367 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 37 1459 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 55 1700 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 64 1777 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 73 1861 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 74 1877 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 86 1972 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii  3  684 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii  6  720 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii  7  726 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 19 1079 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 21 1123 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 23 1188 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 33 1417 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 34 1431 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 38 1496 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 40 1523 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 42 1640 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 50 1666 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 51 1668 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 52 1695 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 65 1781 11A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 44 1649 12A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 49A 1658.1 12A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 49B 1658.2 12A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 56 1707 12A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 80 1893 12A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii  5  693 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii  8  749 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 10  839 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 14  865 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 16  888 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 29 1326 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 35 1440 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 41 1524 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 46 1652 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 47 1653 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 48 1657 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 57 1709 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 61 1727 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 63 1762 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 67 1806 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 75 1881 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 77 1886 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii  1  649 46A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii  2  653 46A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 39 1497 16A29G19C26T21 A31G35C29T34 A40G34C29T36 A. baumannii 24 1198 15A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 28 1243 15A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 43 1648 15A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 62 1746 15A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii  4  689 15A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 68 1822 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 69 1823A 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 70 1823B 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 71 1826 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 72 1860 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 81 1924 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 82 1929 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 85 1966 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 11  841 3A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 32 1415 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 45 1651 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 54 1697 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 58 1712 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 60 1725 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 66 1802 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 76 1883 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 78 1891 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 79 1892 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 83 1947 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 84 1964 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 53 1696 24A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 36 1458 49A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 59 1716 9A27G21C25T22 A32G35C28T34 A39G33C30T37 A. baumannii  9  805 30A27G21C25T22 A32G35C28T34 A39G33C30T37 A. baumannii 18  967 39A27G21C26T21 A32G35C28T34 A39G33C30T37 A. baumannii 30 1322 48A28G21C24T22 A32G35C29T33 A40G33C30T36 A. baumannii 26 1218 50A27G21C25T22 A31G36C28T34 A40G33C29T37 A. sp. 13TU 15  875 A1A27G21C25T22 A30G36C26T37 A41G34C28T36 A. sp. 13TU 17  895 A1A27G21C25T22 A30G36C26T37 A41G34C28T36 A. sp. 3 12  853 B7 A26G23C23T23A30G36C27T36 A39G37C26T37 A. johnsonii 25 1202 NEW1 A25G23C24T23A30G35C30T34 A38G37C26T38 A. sp. 2082 87 2082 NEW2 A26G22C24T23A31G35C28T35 A42G34C27T36

TABLE 17C Base Compositions Determined from A. baumannii DNA SamplesObtained from Walter Reed Hospital and Amplified with TriangulationGenotyping Analysis Primer Pair Nos. 1167 and 1170 and 1171 PP No: 1167PP No: 1170 PP No: 1171 Species Ibis# Isolate ST fumC fumC ppa A.baumannii 20 1082 1 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii13  854 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 22 116210 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 27 1230 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 31 1367 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 37 1459 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 55 1700 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 64 1777 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 73 1861 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 74 1877 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 86 1972 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii  3  684 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii  6  720 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii  7  726 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 19 1079 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 21 1123 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 23 1188 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 33 1417 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 34 1431 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 38 1496 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 40 1523 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 42 1640 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 50 1666 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 51 1668 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 52 1695 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 65 1781 11A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 44 1649 12A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 49A 1658.1 12A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 49B 1658.2 12A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 56 1707 12A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 80 1893 12A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii  5  693 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii  8  749 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 10  839 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 14  865 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 16  888 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 29 1326 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 35 1440 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 41 1524 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 46 1652 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 47 1653 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 48 1657 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 57 1709 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 61 1727 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 63 1762 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 67 1806 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 75 1881 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 77 1886 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii  1  649 46A41G35C32T39 A37G28C20T51 A35G37C32T45 A. baumannii  2  653 46A41G35C32T39 A37G28C20T51 A35G37C32T45 A. baumannii 39 1497 16A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 24 1198 15A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 28 1243 15A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 43 1648 15A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 62 1746 15A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii  4  689 15A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 68 1822 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 69 1823A 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 70 1823B 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 71 1826 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 72 1860 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 81 1924 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 82 1929 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 85 1966 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 11  841 3A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 32 1415 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 45 1651 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 54 1697 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 58 1712 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 60 1725 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 66 1802 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 76 1883 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 78 1891 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 79 1892 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 83 1947 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 84 1964 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 53 1696 24A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 36 1458 49A40G35C34T38 A39G26C22T49 A35G37C30T47 A. baumannii 59 1716 9A40G35C32T40 A38G27C20T51 A36G35C31T47 A. baumannii  9  805 30A40G35C32T40 A38G27C21T50 A35G36C29T49 A. baumannii 18  967 39A40G35C33T39 A38G27C20T51 A35G37C30T47 A. baumannii 30 1322 48A40G35C35T37 A38G27C21T50 A35G37C30T47 A. baumannii 26 1218 50A40G35C34T38 A38G27C21T50 A35G37C33T44 A. sp. 13TU 15  875 A1A41G39C31T36 A37G26C24T49 A34G38C31T46 A. sp. 13TU 17  895 A1A41G39C31T36 A37G26C24T49 A34G38C31T46 A. sp. 3 12  853 B7 A43G37C30T37A36G27C24T49 A34G37C31T47 A. johnsonii 25 1202 NEW1 A42G38C31T36A40G27C19T50 A35G37C32T45 A. sp. 2082 87 2082 NEW2 A43G37C32T35A37G26C21T52 A35G38C31T45

TABLE 18A Base Compositions Determined from A. baumannii DNA SamplesObtained from Northwestern Medical Center and Amplified with SpeciatingPrimer Pair No. 2922 and Triangulation Genotyping Analysis Primer PairNos. 1151 and 1156 PP No: 2922 PP No: 1151 PP No: 1156 Species Ibis#Isolate ST efp trpE adk A. baumannii 54 536 3 A44G35C24T44 A44G35C22T41A44G32C26T38 A. baumannii 87 665 3 A44G35C24T44 A44G35C22T41A44G32C26T38 A. baumannii 8 80 10 A45G34C25T43 A44G35C21T42 A44G32C26T38A. baumannii 9 91 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii10 92 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 11 131 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 12 137 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 21 218 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 26 242 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 94 678 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 1 9 10 A45G34C25T43A44G35C21T42 A44G32C26T38 A. baumannii 2 13 10 A45G34C25T43 A44G35C21T42A44G32C26T38 A. baumannii 3 19 10 A45G34C25T43 A44G35C21T42 A44G32C26T38A. baumannii 4 24 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii5 36 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 6 39 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 13 139 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 15 165 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 16 170 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 17 186 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 20 202 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 22 221 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 24 234 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 25 239 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 33 370 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 34 389 10A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 19 201 14A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 27 257 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 29 301 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 31 354 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 36 422 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 37 424 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 38 434 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 39 473 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 40 482 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 44 512 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 45 516 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 47 522 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 48 526 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 50 528 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 52 531 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 53 533 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 56 542 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 59 550 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 62 556 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 64 557 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 70 588 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 73 603 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 74 605 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 75 606 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 77 611 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 79 622 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 83 643 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 85 653 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 89 669 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 93 674 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 23 228 51A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 32 369 52A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 35 393 52A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 30 339 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 41 485 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 42 493 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 43 502 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 46 520 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 49 527 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 51 529 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 65 562 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 68 579 53A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 57 546 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 58 548 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 60 552 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 61 555 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 63 557 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 66 570 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 67 578 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 69 584 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 71 593 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 72 602 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 76 609 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 78 621 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 80 625 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 81 628 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 82 632 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 84 649 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 86 655 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 88 668 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 90 671 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 91 672 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 92 673 54A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 18 196 55A44G35C25T43 A44G35C20T43 A44G32C27T37 A. baumannii 55 537 27A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 28 263 27A44G35C25T43 A44G35C19T44 A44G32C27T37 A. sp. 3 14 164 B7 A46G35C24T42A42G34C20T46 A43G33C24T40 mixture 7 71 ? mixture ND ND

TABLE 18B Base Compositions Determined from A. baumannii DNA SamplesObtained from Northwestern Medical Center and Amplified withTriangulation Genotyping Analysis Primer Pair Nos. 1158, 1160 and 1165PP No: 1158 PP No: 1160 PP No: 1165 Species Ibis# Isolate ST mutY mutYfumC A. baumannii 54 536 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A.baumannii 87 665 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 880 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 9 91 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 10 92 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 11 131 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 12 137 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 21 218 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 26 242 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 94 678 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 1 9 10 A27G21C26T21A32G35C28T34 A40G33C30T36 A. baumannii 2 13 10 A27G21C26T21 A32G35C28T34A40G33C30T36 A. baumannii 3 19 10 A27G21C26T21 A32G35C28T34 A40G33C30T36A. baumannii 4 24 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii5 36 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 6 39 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 13 139 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 15 165 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 16 170 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 17 186 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 20 202 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 22 221 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 24 234 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 25 239 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 33 370 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 34 389 10A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 19 201 14A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 27 257 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 29 301 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 31 354 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 36 422 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 37 424 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 38 434 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 39 473 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 40 482 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 44 512 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 45 516 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 47 522 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 48 526 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 50 528 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 52 531 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 53 533 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 56 542 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 59 550 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 62 556 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 64 557 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 70 588 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 73 603 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 74 605 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 75 606 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 77 611 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 79 622 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 83 643 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 85 653 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 89 669 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 93 674 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 23 228 51A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 32 369 52A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 35 393 52A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 30 339 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 41 485 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 42 493 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 43 502 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 46 520 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 49 527 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 51 529 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 65 562 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 68 579 53A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 57 546 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 58 548 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 60 552 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 61 555 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 63 557 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 66 570 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 67 578 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 69 584 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 71 593 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 72 602 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 76 609 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 78 621 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 80 625 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 81 628 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 82 632 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 84 649 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 86 655 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 88 668 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 90 671 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 91 672 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 92 673 54A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 18 196 55A27G21C25T22 A31G36C27T35 A40G33C29T37 A. baumannii 55 537 27A27G21C25T22 A32G35C28T34 A40G33C30T36 A. baumannii 28 263 27A27G21C25T22 A32G35C28T34 A40G33C30T36 A. sp. 3 14 164 B7 A26G23C23T23A30G36C27T36 A39G37C26T37 mixture 7 71 ? ND ND ND

TABLE 18C Base Compositions Determined from A. baumannii DNA SamplesObtained from Northwestern Medical Center and Amplified withTriangulation Genotyping Analysis Primer Pair Nos. 1167, 1170 and 1171PP No: 1167 PP No: 1170 PP No: 1171 Species Ibis# Isolate ST fumC fumCppa A. baumannii 54 536 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A.baumannii 87 665 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 880 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 9 91 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 10 92 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 11 131 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 12 137 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 21 218 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 26 242 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 94 678 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 1 9 10 A41G34C34T38A38G27C21T50 A35G37C33T44 A. baumannii 2 13 10 A41G34C34T38 A38G27C21T50A35G37C33T44 A. baumannii 3 19 10 A41G34C34T38 A38G27C21T50 A35G37C33T44A. baumannii 4 24 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii5 36 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 6 39 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 13 139 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 15 165 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 16 170 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 17 186 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 20 202 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 22 221 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 24 234 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 25 239 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 33 370 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 34 389 10A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 19 201 14A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 27 257 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 29 301 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 31 354 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 36 422 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 37 424 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 38 434 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 39 473 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 40 482 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 44 512 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 45 516 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 47 522 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 48 526 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 50 528 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 52 531 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 53 533 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 56 542 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 59 550 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 62 556 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 64 557 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 70 588 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 73 603 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 74 605 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 75 606 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 77 611 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 79 622 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 83 643 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 85 653 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 89 669 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 93 674 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 23 228 51A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 32 369 52A40G35C34T38 A38G27C21T50 A35G37C31T46 A. baumannii 35 393 52A40G35C34T38 A38G27C21T50 A35G37C31T46 A. baumannii 30 339 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 41 485 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 42 493 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 43 502 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 46 520 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 49 527 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 51 529 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 65 562 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 68 579 53A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 57 546 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 58 548 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 60 552 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 61 555 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 63 557 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 66 570 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 67 578 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 69 584 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 71 593 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 72 602 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 76 609 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 78 621 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 80 625 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 81 628 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 82 632 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 84 649 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 86 655 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 88 668 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 90 671 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 91 672 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 92 673 54A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 18 196 55A42G34C33T38 A38G27C20T51 A35G37C31T46 A. baumannii 55 537 27A40G35C33T39 A38G27C20T51 A35G37C33T44 A. baumannii 28 263 27A40G35C33T39 A38G27C20T51 A35G37C33T44 A. sp. 3 14 164 B7 A43G37C30T37A36G27C24T49 A34G37C31T47 mixture 7 71 — ND ND ND

Base composition analysis of the samples obtained from Walter Reedhospital indicated that a majority of the strain types identified werethe same strain types already characterized by the OIF study of Example12. This is not surprising since at least some patients from whichclinical samples were obtained in OIF were transferred to the WalterReed Hospital (WRAIR). Examples of these common strain types include:ST10, ST11, ST12, ST14, ST15, ST16 and ST46. A strong correlation wasnoted between these strain types and the presence of mutations in thegyrA and parC which confer quinolone drug resistance.

In contrast, the results of base composition analysis of samplesobtained from Northwestern Medical Center indicate the presence of 4major strain types: ST10, ST51, ST53 and ST54. All of these strain typeshave the gyrA quinolone resistance mutation and most also have the parCquinolone resistance mutation, with the exception of ST35. Thisobservation is consistent with the current understanding that the gyrAmutation generally appears before the parC mutation and suggests thatthe acquisition of these drug resistance mutations is rather recent andthat resistant isolates are taking over the wild-type isolates. Anotherinteresting observation was that a single isolate of ST3 (isolate 841)displays a triangulation genotyping analysis pattern similar to otherisolates of ST3, but the codon analysis amplification product basecompositions indicate that this isolate has not yet undergone thequinolone resistance mutations in gyrA and parC.

The six isolates that represent species other than Acinetobacterbaumannii in the samples obtained from the Walter Reed Hospital wereeach found to not carry the drug resistance mutations.

The results described above involved analysis of 183 samples using themethods and compositions of the present invention. Results were providedto collaborators at the Walter Reed hospital and Northwestern Medicalcenter within a week of obtaining samples. This example highlights therapid throughput characteristics of the analysis platform and theresolving power of triangulation genotyping analysis and codon analysisfor identification of and determination of drug resistance in bacteria.

Example 14 Identification of Drug Resistance Genes and Virulence Factorsin Staphylococcus aureus

An eight primer pair panel was designed for identification of drugresistance genes and virulence factors of Staphylococcus aureus and isshown in Table 19. The primer sequences are found in Table 2 and arecross-referenced by the primer pair numbers, primer pair names or SEQ IDNOs listed in Table 19. TABLE 19 Primer Pairs for Identification of DrugResistance Genes and Virulence Factors in Staphylococcus aureus ForwardReverse Primer Primer Primer Pair (SEQ ID (SEQ ID Target No. ForwardPrimer Name NO:) Reverse Primer Name NO:) Gene 879MECA_Y14051_4507_4530_F 288 MECA_Y14051_4555_4581_R 1269 mecA 2056MECI-R_NC003923-41798- 698 MECI-R_NC003923-41798- 1420 MecI-R41609_33_60_F 41609_86_113_R 2081 ERMA_NC002952-55890- 217ERMA_NC002952-55890- 1167 ermA 56621_366_395_F 56621_438_465_R 2086ERMC_NC005908-2004- 399 ERMC_NC005908-2004- 1041 ermC 2738_85_116_F2738_173_206_R 2095 PVLUK_NC003923-1529595- 456 PVLUK_NC003923-1529595-1261 Pv-luk 1531285_688_713_F 1531285_775_804_R 2249TUFB_NC002758-615038- 430 TUFB_NC002758-615038- 1321 tufB616222_696_725_F 616222_793_820_R 2256 NUC_NC002758-894288- 174NUC_NC002758-894288- 853 Nuc 894974_316_345_F 894974_396_421_R 2313MUPR_X75439_2486_2516_F 172 MUPR_X75439_2548_2574_R 1360 mupR

Primer pair numbers 2256 and 2249 are confirmation primers designed withthe aim of high level identification of Staphylococcus aureus. The nucgene is a Staphylococcus aureus-specific marker gene. The tufB gene is auniversal housekeeping gene but the bioagent identifying amplicondefined by primer pair number 2249 provides a unique base composition(A43 G28 C19 T35) which distinguishes Staphylococcus aureus from othermembers of the genus Staphylococcus.

High level methicillin resistance in a given strain of Staphylococcusaureus is indicated by bioagent identifying amplicons defined by primerpair numbers 879 and 2056. Analyses have indicated that primer pairnumber 879 is not expected to prime S. sciuri homolog or Enterococcusfaecalis/faciem ampicillin-resistant PBP5 homologs.

Macrolide and erythromycin resistance in a given strain ofStaphylococcus aureus is indicated by bioagent identifying ampliconsdefined by primer pair numbers 2081 and 2086.

Resistance to mupriocin in a given strain of Staphylococcus aureus isindicated by bioagent identifying amplicons defined by primer pairnumber 2313.

Virulence in a given strain of Staphylococcus aureus is indicated bybioagent identifying amplicons defined by primer pair number 2095. Thisprimer pair can simultaneously and identify the pvl (lukS-PV) gene andthe lukD gene which encodes a homologous enterotoxin. A bioagentidentifying amplicon of the lukD gene has a six nucleobase lengthdifference relative to the lukS-PV gene.

A total of 32 blinded samples of different strains of Staphylococcusaureus were provided by the Center for Disease Control (CDC). Eachsample was analyzed by PCR amplification with the eight primer pairpanel, followed by purification and measurement of molecular masses ofthe amplification products by mass spectrometry. Base compositions forthe amplification products were calculated. The base compositionsprovide the information summarized above for each primer pair. Theresults are shown in Tables 20A and B. One result noted upon un-blindingof the samples is that each of the PVL+ identifications agreed with PVL+identified in the same samples by standard PCR assays. These resultsindicate that the panel of eight primer pairs is useful foridentification of drug resistance and virulence sub-speciescharacteristics for Staphylococcus aureus. It is expected that a kitcomprising one or more of the members of this panel will be a usefulembodiment of the present invention. TABLE 20A Drug Resistance andVirulence Identified in Blinded Samples of Various Strains ofStaphylococcus aureus with Primer Pair Nos. 2081, 2086, 2095 and 2256Primer Primer Pair No. Pair No. 2081 2086 Primer Pair No. Primer PairNo. Sample Index No. (ermA) (ermC) 2095 (pv-luk) 2256 (nuc) CDC0010 − −PVL−/lukD+ + CDC0015 − − PVL+/lukD+ + CDC0019 − + PVL−/lukD+ + CDC0026 +− PVL−/lukD+ + CDC0030 + − PVL−/lukD+ + CDC004 − − PVL+/lukD+ + CDC0014− + PVL+/lukD+ + CDC008 − − PVL−/lukD+ + CDC001 + − PVL−/lukD+ +CDC0022 + − PVL−/lukD+ + CDC006 + − PVL−/lukD+ + CDC007 − − PVL−/lukD+ +CDCVRSA1 + − PVL−/lukD+ + CDCVRSA2 + + PVL−/lukD+ + CDC0011 + −PVL−/lukD+ + CDC0012 − − PVL+/lukD− + CDC0021 + − PVL−/lukD+ + CDC0023 +− PVL−/lukD+ + CDC0025 + − PVL−/lukD+ + CDC005 − − PVL−/lukD+ +CDC0018 + − PVL+/lukD− + CDC002 − − PVL−/lukD+ + CDC0028 + −PVL−/lukD+ + CDC003 − − PVL−/lukD+ + CDC0013 − − PVL+/lukD+ + CDC0016 −− PVL−/lukD+ + CDC0027 + − PVL−/lukD+ + CDC0029 − − PVL+/lukD+ + CDC0020− + PVL−/lukD+ + CDC0024 − − PVL−/lukD+ + CDC0031 − − PVL−/lukD+ +

TABLE 20B Drug Resistance and Virulence Identified in Blinded Samples ofVarious Strains of Staphylococcus aureus with Primer Pair Nos. 2249,879, 2056, and 2313 Primer Primer Primer Pair No. Pair No. Pair No.Sample Primer Pair No. 2249 879 2056 2313 Index No. (tufB) (mecA)(mecI-R) (mupR) CDC0010 Staphylococcus aureus + + − CDC0015Staphylococcus aureus − − − CDC0019 Staphylococcus aureus + + − CDC0026Staphylococcus aureus + + − CDC0030 Staphylococcus aureus + + − CDC004Staphylococcus aureus + + − CDC0014 Staphylococcus aureus + + − CDC008Staphylococcus aureus + + − CDC001 Staphylococcus aureus + + − CDC0022Staphylococcus aureus + + − CDC006 Staphylococcus aureus + + + CDC007Staphylococcus aureus + + − CDCVRSA1 Staphylococcus aureus + + −CDCVRSA2 Staphylococcus aureus + + − CDC0011 Staphylococcus aureus − − −CDC0012 Staphylococcus aureus + + − CDC0021 Staphylococcus aureus + + −CDC0023 Staphylococcus aureus + + − CDC0025 Staphylococcus aureus + + −CDC005 Staphylococcus aureus + + − CDC0018 Staphylococcus aureus + + −CDC002 Staphylococcus aureus + + − CDC0028 Staphylococcus aureus + + −CDC003 Staphylococcus aureus + + − CDC0013 Staphylococcus aureus + + −CDC0016 Staphylococcus aureus + + − CDC0027 Staphylococcus aureus + + −CDC0029 Staphylococcus aureus + + − CDC0020 Staphylococcus aureus − − −CDC0024 Staphylococcus aureus + + − CDC0031 Staphylococcus scleiferi − −−

Example 15 Selection and Use of Triangulation Genotyping Analysis PrimerPairs for Staphylococcus aureus

To combine the power of high-throughput mass spectrometric analysis ofbioagent identifying amplicons with the sub-species characteristicresolving power provided by triangulation genotyping analysis, a panelof eight triangulation genotyping analysis primer pairs was selected.The primer pairs are designed to produce bioagent identifying ampliconswithin six different housekeeping genes which are listed in Table 21.The primer sequences are found in Table 2 and are cross-referenced bythe primer pair numbers, primer pair names or SEQ ID NOs listed in Table21. TABLE 21 Primer Pairs for Triangulation Genotyping Analysis ofStaphylococcus aureus Forward Reverse Primer Primer Primer Pair (SEQ ID(SEQ ID Target No. Forward Primer Name NO:) Reverse Primer Name NO:)Gene 2146 ARCC_NC003923-2725050- 437 ARCC_NC003923-2725050- 1137 arcC2724595_131_161_F 2724595_214_245_R 2149 AROE_NC003923-1674726- 530AROE_NC003923-1674726- 891 aroE 1674277_30_62_F 1674277_155_181_R 2150AROE_NC003923-1674726- 474 AROE_NC003923-1674726- 869 aroE1674277_204_232_F 1674277_308_335_R 2156 GMK_NC003923-1190906- 268GMK_NC003923-1190906- 1284 gmk 1191334_301_329_F 1191334_403_432_R 2157PTA_NC003923-628885- 418 PTA_NC003923-628885- 1301 pta 629355_237_263_F629355_314_345_R 2161 TPI_NC003923-830671- 318 TPI_NC003923-830671- 1300tpi 831072_1_34_F 831072_97_129_R 2163 YQI_NC003923-378916- 440YQI_NC003923-378916- 1076 yqi 379431_142_167_F 379431_259_284_R 2166YQI_NC003923-378916- 219 YQI_NC003923-378916- 1013 yqi 379431_275_300_F379431_364_396_R

The same samples analyzed for drug resistance and virulence in Example14 were subjected to triangulation genotyping analysis. The primer pairsof Table 21 were used to produce amplification products by PCR, whichwere subsequently purified and measured by mass spectrometry. Basecompositions were calculated from the molecular masses and are shown inTables 22A and 22B. TABLE 22A Triangulation Genotyping Analysis ofBlinded Samples of Various Strains of Staphylococcus aureus with PrimerPair Nos. 2146, 2149, 2150 and 2156 Sample Primer Pair No. Primer PairNo. Primer Pair No. Primer Pair No. Index No. Strain 2146 (arcC) 2149(aroE) 2150 (aroE) 2156 (gmk) CDC0010 COL A44 G24 C18 T29 A59 G24 C18T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC0015 COL A44 G24 C18 T29 A59 G24C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC0019 COL A44 G24 C18 T29 A59G24 C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC0026 COL A44 G24 C18 T29A59 G24 C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC0030 COL A44 G24 C18T29 A59 G24 C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC004 COL A44 G24C18 T29 A59 G24 C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC0014 COL A44G24 C18 T29 A59 G24 C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC008 ????A44 G24 C18 T29 A59 G24 C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC001Mu50 A45 G23 C20 T27 A58 G24 C18 T52 A40 G36 C13 T43 A51 G29 C21 T31CDC0022 Mu50 A45 G23 C20 T27 A58 G24 C18 T52 A40 G36 C13 T43 A51 G29 C21T31 CDC006 Mu50 A45 G23 C20 T27 A58 G24 C18 T52 A40 G36 C13 T43 A51 G29C21 T31 CDC0011 MRSA252 A45 G24 C18 T28 A58 G24 C19 T51 A41 G36 C12 T43A51 G29 C21 T31 CDC0012 MRSA252 A45 G24 C18 T28 A58 G24 C19 T51 A41 G36C12 T43 A51 G29 C21 T31 CDC0021 MRSA252 A45 G24 C18 T28 A58 G24 C19 T51A41 G36 C12 T43 A51 G29 C21 T31 CDC0023 ST: 110 A45 G24 C18 T28 A59 G24C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC0025 ST: 110 A45 G24 C18 T28A59 G24 C18 T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC005 ST: 338 A44 G24C18 T29 A59 G23 C19 T51 A40 G36 C14 T42 A51 G29 C21 T31 CDC0018 ST: 338A44 G24 C18 T29 A59 G23 C19 T51 A40 G36 C14 T42 A51 G29 C21 T31 CDC002ST: 108 A46 G23 C20 T26 A58 G24 C19 T51 A42 G36 C12 T42 A51 G29 C20 T32CDC0028 ST: 108 A46 G23 C20 T26 A58 G24 C19 T51 A42 G36 C12 T42 A51 G29C20 T32 CDC003 ST: 107 A45 G23 C20 T27 A58 G24 C18 T52 A40 G36 C13 T43A51 G29 C21 T31 CDC0013 ST: 12 ND A59 G24 C18 T51 A40 G36 C13 T43 A51G29 C21 T31 CDC0016 ST: 120 A45 G23 C18 T29 A58 G24 C19 T51 A40 G37 C13T42 A51 G29 C21 T31 CDC0027 ST: 105 A45 G23 C20 T27 A58 G24 C18 T52 A40G36 C13 T43 A51 G29 C21 T31 CDC0029 MSSA476 A45 G23 C20 T27 A58 G24 C19T51 A40 G36 C13 T43 A50 G30 C20 T32 CDC0020 ST: 15 A44 G23 C21 T27 A59G23 C18 T52 A40 G36 C13 T43 A50 G30 C20 T32 CDC0024 ST: 137 A45 G23 C20T27 A57 G25 C19 T51 A40 G36 C13 T43 A51 G29 C22 T30 CDC0031 *** Noproduct No product No product No product

TABLE 22B Triangulation Genotyping Analysis of Blinded Samples ofVarious Strains of Staphylococcus aureus with Primer Pair Nos. 2146,2149, 2150 and 2156 Sample Primer Pair No. Primer Pair No. Primer PairNo. Primer Pair No. Index No. Strain 2157 (pta) 2161 (tpi) 2163 (yqi)2166 (yqi) CDC0010 COL A32 G25 C23 T29 A51 G28 C22 T28 A41 G37 C22 T43A37 G30 C18 T37 CDC0015 COL A32 G25 C23 T29 A51 G28 C22 T28 A41 G37 C22T43 A37 G30 C18 T37 CDC0019 COL A32 G25 C23 T29 A51 G28 C22 T28 A41 G37C22 T43 A37 G30 C18 T37 CDC0026 COL A32 G25 C23 T29 A51 G28 C22 T28 A41G37 C22 T43 A37 G30 C18 T37 CDC0030 COL A32 G25 C23 T29 A51 G28 C22 T28A41 G37 C22 T43 A37 G30 C18 T37 CDC004 COL A32 G25 C23 T29 A51 G28 C22T28 A41 G37 C22 T43 A37 G30 C18 T37 CDC0014 COL A32 G25 C23 T29 A51 G28C22 T28 A41 G37 C22 T43 A37 G30 C18 T37 CDC008 unknown A32 G25 C23 T29A51 G28 C22 T28 A41 G37 C22 T43 A37 G30 C18 T37 CDC001 Mu50 A33 G25 C22T29 A50 G28 C22 T29 A42 G36 C22 T43 A36 G31 C19 T36 CDC0022 Mu50 A33 G25C22 T29 A50 G28 C22 T29 A42 G36 C22 T43 A36 G31 C19 T36 CDC006 Mu50 A33G25 C22 T29 A50 G28 C22 T29 A42 G36 C22 T43 A36 G31 C19 T36 CDC0011MRSA252 A32 G25 C23 T29 A50 G28 C22 T29 A42 G36 C22 T43 A37 G30 C18 T37CDC0012 MRSA252 A32 G25 C23 T29 A50 G28 C22 T29 A42 G36 C22 T43 A37 G30C18 T37 CDC0021 MRSA252 A32 G25 C23 T29 A50 G28 C22 T29 A42 G36 C22 T43A37 G30 C18 T37 CDC0023 ST: 110 A32 G25 C23 T29 A51 G28 C22 T28 A41 G37C22 T43 A37 G30 C18 T37 CDC0025 ST: 110 A32 G25 C23 T29 A51 G28 C22 T28A41 G37 C22 T43 A37 G30 C18 T37 CDC005 ST: 338 A32 G25 C24 T28 A51 G27C21 T30 A42 G36 C22 T43 A37 G30 C18 T37 CDC0018 ST: 338 A32 G25 C24 T28A51 G27 C21 T30 A42 G36 C22 T43 A37 G30 C18 T37 CDC002 ST: 108 A33 G25C23 T28 A50 G28 C22 T29 A42 G36 C22 T43 A37 G30 C18 T37 CDC0028 ST: 108A33 G25 C23 T28 A50 G28 C22 T29 A42 G36 C22 T43 A37 G30 C18 T37 CDC003ST: 107 A32 G25 C23 T29 A51 G28 C22 T28 A41 G37 C22 T43 A37 G30 C18 T37CDC0013 ST: 12 A32 G25 C23 T29 A51 G28 C22 T28 A42 G36 C22 T43 A37 G30C18 T37 CDC0016 ST: 120 A32 G25 C24 T28 A50 G28 C21 T30 A42 G36 C22 T43A37 G30 C18 T37 CDC0027 ST: 105 A33 G25 C22 T29 A50 G28 C22 T29 A43 G36C21 T43 A36 G31 C19 T36 CDC0029 MSSA476 A33 G25 C22 T29 A50 G28 C22 T29A42 G36 C22 T43 A36 G31 C19 T36 CDC0020 ST: 15 A33 G25 C22 T29 A50 G28C21 T30 A42 G36 C22 T43 A36 G31 C18 T37 CDC0024 ST: 137 A33 G25 C22 T29A51 G28 C22 T28 A42 G36 C22 T43 A37 G30 C18 T37 CDC0031 *** A34 G25 C25T25 A51 G27 C24 T27 No product No productNote:*** The sample CDC0031 was identified as Staphylococcus scleiferi asindicated in Example 14. Thus, the triangulation genotyping primersdesigned for Staphylococcus aureus would# generally not be expected to prime and produce amplification productsof this organism. Tables 22A and 22B indicate that amplificationproducts are obtained for this organism only with primer pair numbers2157 and 2161.

A total of thirteen different genotypes of Staphylococcus aureus wereidentified according to the unique combinations of base compositionsacross the eight different bioagent identifying amplicons obtained withthe eight primer pairs. These results indicate that this eight primerpair panel is useful for analysis of unknown or newly emerging strainsof Staphylococcus aureus. It is expected that a kit comprising one ormore of the members of this panel will be a useful embodiment of thepresent invention.

Example 16 Selection and Use of Triangulation Genotyping Analysis PrimerPairs for Members of the Bacterial Genus Vibrio

To combine the power of high-throughput mass spectrometric analysis ofbioagent identifying amplicons with the sub-species characteristicresolving power provided by triangulation genotyping analysis, a panelof eight triangulation genotyping analysis primer pairs was selected.The primer pairs are designed to produce bioagent identifying ampliconswithin seven different housekeeping genes which are listed in Table 23.The primer sequences are found in Table 2 and are cross-referenced bythe primer pair numbers, primer pair names or SEQ ID NOs listed in Table23. TABLE 23 Primer Pairs for Triangulation Genotyping Analysis ofMembers of the Bacterial Genus Vibrio Forward Reverse Primer PrimerPrimer Pair (SEQ ID (SEQ ID Target No. Forward Primer Name NO:) ReversePrimer Name NO:) Gene 1098 RNASEP_VBC_331_349_F 325 RNASEP_VBC_388_414_R1163 RNAse P 2000 CTXB_NC002505_46_70_F 278 CTXB_NC002505_132_162_R 1039ctxB 2001 FUR_NC002505_87_113_F 465 FUR_NC002505_205_228_R 1037 fur 2011GYRB_NC002505_1161_1190_F 148 GYRB_NC002505_1255_1284_R 1172 gyrB 2012OMPU_NC002505_85_110_F 190 OMPU_NC002505_154_180_R 1254 ompU 2014OMPU_NC002505_431_455_F 266 OMPU_NC002505_544_567_R 1094 ompU 2323CTXA_NC002505-1568114- 508 CTXA_NC002505-1568114- 1297 ctxA1567341_122_149_F 1567341_186_214_R 2927 GAPA_NC002505_694_721_F 259GAPA_NC_002505_29_58_R 1060 gapA

A group of 50 bacterial isolates containing multiple strains of bothenvironmental and clinical isolates of Vibrio cholerae, 9 other Vibniospecies, and 3 species of Photobacteria were tested using this panel ofprimer pairs. Base compositions of amplification products obtained withthese 8 primer pairs were used to distinguish amongst various speciestested, including sub-species differentiation within Vibrio choleraeisolates. For instance, the non-O1/non-O139 isolates were clearlyresolved from the O1 and the O139 isolates, as were several of theenvironmental isolates of Vibrio cholerae from the clinical isolates.

It is expected that a kit comprising one or more of the members of thispanel will be a useful embodiment of the present invention.

Example 17 Selection and Use of Triangulation Genotyping Analysis PrimerPairs for Members of the Bacterial Genus Pseudomonas

To combine the power of high-throughput mass spectrometric analysis ofbioagent identifying amplicons with the sub-species characteristicresolving power provided by triangulation genotyping analysis, a panelof twelve triangulation genotyping analysis primer pairs was selected.The primer pairs are designed to produce bioagent identifying ampliconswithin seven different housekeeping genes which are listed in Table 24.The primer sequences are found in Table 2 and are cross-referenced bythe primer pair numbers, primer pair names or SEQ ID NOs listed in Table24. TABLE 24 Primer Pairs for Triangulation Genotyping Analysis ofMembers of the Bacterial Genus Pseudomonas Forward Reverse Primer PrimerPrimer Pair (SEQ ID (SEQ ID Target No. Forward Primer Name NO:) ReversePrimer Name NO:) Gene 2949 ACS_NC002516-970624- 376 ACS_NC002516-970624-1265 acsA 971013_299_316_F 971013_364_383_R 2950 ARO_NC002516-26883- 267ARO_NC002516-26883- 1341 aroE 27380_4_26_F 27380_111_128_R 2951ARO_NC002516-26883- 705 ARO_NC002516-26883- 1056 aroE 27380_356_377_F27380_459_484_R 2954 GUA_NC002516-4226546- 710 GUA_NC002516-4226546-1259 guaA 4226174_155_178_F 4226174_265_287_R 2956 GUA_NC002516-4226546-374 GUA_NC002516-4226546- 1111 guaA 4226174_242_263_F 4226174_355_371_R2957 MUT_NC002516-5551158- 545 MUT_NC002516-5551158-  978 mutL5550717_5_26_F 5550717_99_116_R 2959 NUO_NC002516-2984589- 249NUO_NC002516-2984589- 1095 nuoD 2984954_8_26_F 2984954_97_117_R 2960NUO_NC002516-2984589- 195 NUO_NC002516-2984589- 1376 nuoD2984954_218_239_F 2984954_301_326_R 2961 PPS_NC002516-1915014- 311PPS_NC002516-1915014- 1014 pps 1915383_44_63_F 1915383_140_165_R 2962PPS_NC002516-1915014- 365 PPS_NC002516-1915014- 1052 pps1915383_240_258_F 1915383_341_360_R 2963 TRP_NC002516-671831- 527TRP_NC002516-671831- 1071 trpE 672273_24_42_F 672273_131_150_R 2964TRP_NC002516-671831- 490 TRP_NC002516-671831- 1182 trpE 672273_261_282_F672273_362_383_R

It is expected that a kit comprising one or more of the members of thispanel will be a useful embodiment of the present invention.

The present invention includes any combination of the various speciesand subgeneric groupings falling within the generic disclosure. Thisinvention therefore includes the generic description of the inventionwith a proviso or negative limitation removing any subject matter fromthe genus, regardless of whether or not the excised material isspecifically recited herein.

While in accordance with the patent statutes, description of the variousembodiments and examples have been provided, the scope of the inventionis not to be limited thereto or thereby. Modifications and alterationsof the present invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the present invention.

Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims, rather than by the specific exampleswhich have been presented by way of example.

Each reference (including, but not limited to, journal articles, U.S.and non-U.S. patents, patent application publications, internationalpatent application publications, gene bank gi or accession numbers,internet web sites, and the like) cited in the present application isincorporated herein by reference in its entirety.

1. An oligonucleotide primer pair comprising a forward primer and areverse primer, each comprising between 13 and 35 linked nucleotides inlength, configured to generate an amplicon that is between 45 and 200linked nucleotides in length, said forward primer configured tohybridize with at least 80% complementarity to a first portion of aregion of Genbank gi number: 57634611, and said reverse primerconfigured to hybridize with at least 80% complementarity to a secondportion of said region of Genbank gi number: 57634611, wherein saidregion of Genbank gi number: 57634611 begins with the 5′ end of SEQ IDNO.: 619, and extends to the 5′ end of SEQ ID NO.:
 907. 2. Theoligonucleotide primer pair of claim 1, wherein said forward primercomprises at least 90% complementarity to said first portion of saidregion of Genbank gi number 57634611, and wherein said first portion iswithin a region beginning with the 5′ end of SEQ ID NO: 619 andextending to the 3′ end of SEQ ID NO.:
 304. 3. The oligonucleotideprimer pair of claim 2, wherein said forward primer comprises at least95% complementarity to said first portion.
 4. The oligonucleotide primerpair of claim 3, wherein said forward primer comprises 100%complementarity to said first portion.
 5. The oligonucleotide primerpair of claim 1, wherein said reverse primer comprises at least 90%complementarity to said second portion of said region of Genbank ginumber 57634611, and wherein said second portion is within a regionbeginning with the 3′ end of SEQ ID NO: 1312 and extending to the 5′ endof SEQ ID NO.:
 907. 6. The oligonucleotide primer pair of claim 5,wherein said reverse primer comprises at least 95% complementarity tosaid second portion.
 7. The oligonucleotide primer pair of claim 6,wherein said reverse primer comprises 100% complementarity to saidsecond portion.
 8. The oligonucleotide primer pair of claim 1, whereinsaid forward primer comprises at least 70% sequence identity with SEQ IDNO:
 304. 9. The oligonucleotide primer pair of claim 1, wherein saidforward primer is SEQ ID NO:
 304. 10. The oligonucleotide primer pair ofclaim 1, wherein said reverse primer comprises at least 70% sequenceidentity with SEQ ID NO:
 907. 11. The oligonucleotide primer pair ofclaim 1, wherein said reverse primer is SEQ ID NO:
 907. 12. Theoligonucleotide primer pair of claim 1, wherein at least one of saidforward primer and said reverse primer comprises at least one modifiednucleobase.
 13. The oligonucleotide primer pair of claim 12, wherein atleast one of said at least one modified nucleobase is a mass modifiednucleobase.
 14. The oligonucleotide primer pair of claim 13, whereinsaid mass modified nucleobase is 5-Iodo-C.
 15. The composition of claim13, wherein said mass modified nucleobase comprises a molecular massmodifying tag.
 16. The oligonucleotide primer pair of claim 12, whereinat least one of said at least one modified nucleobase is a universalnucleobase.
 17. The oligonucleotide primer pair of claim 16, whereinsaid universal nucleobase is inosine.
 18. The oligonucleotide primerpair of claim 1, wherein at least one of said forward primer and saidreverse primer comprises a non-templated T residue at its 5′ end.
 19. Akit for identifying a Staphylococcus aureus bioagent comprising: i) afirst oligonucleotide primer pair comprising a forward primer and areverse primer, each comprising between 13 and 35 linked nucleotides inlength, configured to generate an amplicon that is between 45 and 200linked nucleotides in length, said forward primer configured tohybridize with at least 80% complementarity to a first portion of aregion of Genbank gi number: 57634611, and said reverse primerconfigured to hybridize with at least 80% complementarity to a secondportion of said region of Genbank gi number: 57634611, wherein saidregion of Genbank gi number: 57634611 begins with the 5′ end of SEQ IDNO.: 619, and extends to the 5′ end of SEQ ID NO.: 907; and ii) at leastone additional primer pair, wherein the primers of each of said at leastone additional primer pair are designed to hybridize to conservedsequence regions within a Staphylococcus aureus gene selected from thegroup consisting of tsst, mecA, mecR1, ermA, ermC, pvluk, mupR, and nuc.20. The kit of claim 19 wherein each of said at least one additionalprimer pair comprises SEQ ID NO: 217:SEQ ID NO: 1167, SEQ ID NO: 399:SEQID NO: 1041, SEQ ID NO: 456:SEQ ID NO: 1261, SEQ ID NO: 430:SEQ ID NO:1321, SEQ ID NO: 288:SEQ ID NO: 1269, SEQ ID NO: 698:SEQ ID NO: 1420,SEQ ID NO: 205:SEQ ID NO: 876, or SEQ ID NO: 174:SEQ ID NO:
 853. 21. Thekit of claim 19 wherein said first oligonucleotide primer pair and saidat least one additional primer pair consists of eight oligonucleotideprimer pairs having at least 70% sequence identity with the primerpairs: SEQ ID NO: 217:SEQ ID NO: 1167, SEQ ID NO: 399:SEQ ID NO: 1041,SEQ ID NO: 456:SEQ ID NO: 1261, SEQ ID NO: 430:SEQ ID NO: 1321, SEQ IDNO: 288:SEQ ID NO: 1269, SEQ ID NO: 698:SEQ ID NO: 1420, SEQ ID NO:304:SEQ ID NO: 907, and SEQ ID NO: 174:SEQ ID NO:
 853. 22. A method foridentifying a Staphylococcus aureus bioagent in a sample comprising: a)amplifying a nucleic acid from said sample using an oligonucleotideprimer pair comprising a forward primer and a reverse primer, eachcomprising between 13 and 35 linked nucleotides in length, said forwardprimer configured to hybridize with at least 80% complementarity to afirst portion of a region of Genbank gi number: 57634611, and saidreverse primer configured to hybridize with at least 80% complementarityto a second portion of said region of Genbank gi number: 57634611,wherein said region of Genbank gi number: 57634611 begins with the 5′end of SEQ ID NO.: 619, and extends to the 5′ end of SEQ ID NO.: 907,wherein said amplifying generates at least one amplification productthat comprises between 45 and 200 linked nucleotides; and b) determiningthe molecular mass of said at least one amplification product by massspectrometry.
 23. The method of claim 22 further comprising comparingsaid determined molecular mass to a database comprising a plurality ofmolecular masses of bioagent identifying amplicons, wherein a matchbetween said determined molecular mass and a molecular mass comprised insaid database identifies said Staphylococcus aureus bioagent in saidsample.
 24. The method of claim 22 further comprising calculating a basecomposition of said at least one amplification product using saidmolecular mass.
 25. The method of claim 24 further comprising comparingsaid calculated base composition to a database comprising a plurality ofbase compositions of bioagent identifying amplicons, wherein a matchbetween said calculated base composition and a base compositioncomprised in said database identifies said Staphylococcus aureusbioagent in said sample.
 26. The method of claim 22, wherein saidforward primer comprises at least 70% sequence identity with SEQ ID NO:304.
 27. The method of claim 22, wherein said reverse primer comprisesat least 70% sequence identity with SEQ ID NO:
 907. 28. The method ofclaim 22 further comprising repeating said amplifying and determiningsteps using at least one additional oligonucleotide primer pair whereinthe primers of each of said at least one additional primer pair aredesigned to hybridize to conserved sequence regions within aStaphylococcus aureus gene selected from the group consisting of mecA,mecR1, ermA, ermC, pvluk, tufb, mupR, tsst, and nuc.
 29. The method ofclaim 22, wherein said identifying comprises detecting the presence ofsaid Staphylococcus aureus bioagent in said sample.
 30. The method ofclaim 22, wherein said identifying comprises determining either thesensitivity or the resistance of said Staphylococcus aureus bioagent insaid sample to one or more antibiotics.
 31. The method of claim 22,wherein said identifying comprises identifying a sub-speciescharacteristic, strain, or genotype of said Staphylococcus aureusbioagent in said sample.